Nonnucleoside inhibitors of reverse transcriptase, composite binding pocket and methods for use thererof

ABSTRACT

Novel compounds that are potent inhibitors of HIV reverse transcriptase (RT) are described in the invention. The novel compounds also inhibit replication of a retrovirus, such as human immunodeficiency virus-1 (HIV-1). The novel compounds of the invention include analogs and derivatives of phenethylthiazolylthiourea (PETT), of dihydroalkoxybenzyloxopyrimidine (DABO), and of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). 
     The invention additionally provides a composite HIV reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket constructed from a composite of multiple NNI-RT complexes The composite RT-NNI binding pocket provides a unique and useful tool for designing and identifying novel, potent inhibitors of reverse transcriptase.

CROSS-REFERENCE

This application in a continuation of application Ser. No. 09/690,265 filed Oct. 17, 2000 now U.S. Pat. No. 5,998,411 which is a continuation of application Ser. No. 09/205,167 filed Dec. 4, 1998 now U.S. Pat. No. 6,180,654 which is a continuation of application Ser. No. 09/040,538 filed Mar. 17, 1998 now U.S. Pat. No. 5,998,411 which applications are incorporated herein by reference.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

The inventors acknowledge and appreciate the assistance of Dr. Elise Sudbeck.

BACKGROUND OF THE INVENTION

Design of potent inhibitors of human immunodeficiency virus (HIV-1) reverse transcriptase (RT), an enzyme responsible for the reverse transcription of the retroviral RNA to proviral DNA, has been a focal point in translational AIDS research efforts (Greene, W. C., New England Journal of Medicine, 1991, 324, 308-317; Mitsuya, H. et al., Science, 1990, 249, 1533-1544; De Clercq, E., J. Acquired Immune Defic. Syndr. Res. Human. Retrovirus, 1992, 8, 119-134). Promising inhibitors include nonnucleoside inhibitors (NNI), which bind to a specific allosteric site of HIV-1 RT near the polymerase site and interfere with reverse transcription by altering either the conformation or mobility of RT, thereby leading to noncompetitive inhibition of the enzyme (Kohlstaedt, L. A. et al., Science, 1992, 256, 1783-1790).

NNI of HIV-1 RT include the following:

(a) 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT; Tanaka, H. et al., J. Med. Chem., 1991, 34, 349-357; Pontikis, R. et al., J. Med. Chem., 1997, 40, 1845-1854;

Danel, K., et al., J. Med. Chem., 1996, 39, 2427-2431; Baba, M., et al., Antiviral Res, 1992, 17, 245-264);

(b) tetrahydroimidazobenzodiazepinethiones (TIBO; Pauwels, R. et al., Nature, 1990, 343, 470-474);

(c) bis(heteroaryl)piperazines (BHAP; Romero, D. L. et al., J. Med. Chem., 1993, 36, 1505-1508);

(d) dihydroalkoxybenzyloxopyrimidine (DABO; Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med. Chem., 1997, 40, 1447-1454);

e) 2′-5′-bis-O-(tertbutyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2″-dioxide) pyrimidines (TSAO; Balzarini, J. et al., Proc. Natl. Acad. Sci. U S A, 1992, 89, 4392-4396); and

(f) phenethylthiazolylthiourea (PETT) derivatives (Bell, F. W. et al., J. Med. Chem., 1995, 38, 4929-4936; Cantrell, A. S. et al., J. Med. Chem., 1996, 39, 4261-4274).

Current protein structure-based drug design efforts rely heavily on crystal structure information of the target binding site. A number of crystal structures of RT complexed with NNIs (including α-APA, TIBO, Nevirapine, BHAP and HEPT derivatives) have been reported, and such structural information provides the basis for further derivatization of NNI aimed at maximizing binding affinity to RT. However, the number of available crystal structures of RT NNI complexes is limited, and no structural information has been reported for RT-PETT complexes or RT-DABO complexes. Given the lack of structural information, researchers must rely on other design procedures for preparing active PETT and DABO derivatives. One of the first reported strategies for systematic synthesis of PETT derivatives was the analysis of structure-activity relationships independent of the structural properties of RT and led to the development of some PETT derivatives with significant anti-HIV activity (Bell, F. W. et al., J. Med. Chem., 1995, 38, 4929-4936; Cantrell, A. S. et al., J. Med. Chem., 1996, 39, 4261-4274). The inclusion of structural information in the drug design process should lead to more efficient identification of promising RT inhibitors.

Although the crystal structure of an RT-NNI complex can be used to provide useful information for the design of a different type of NNI, its application is limited. For example, an analysis of the RT-APA (α-anilinophenylacetamide) complex structure would not predict that the chemically dissimilar inhibitor TNK (6-benzyl-1-benzyloxymethyl uracil) could bind in the same region. The RT-APA structure reveals that there would not be enough room in the APA binding site for the 1-benzyloxymethyl group of TNK (Hopkins, A. L. et al., J. Med. Chem., 1996, 39, 1589-1600). Nevertheless TNK is known to bind in this region as evidenced by the crystal structure of RT-TNK which shows that RT residues can adjust to accommodate the 1-benzyloxymethyl group. Conversely, an analysis of the RT-TNK complex would not predict favorable binding of APA in the TNK binding site. The structure does not show how residue E138 can move to accommodate the 2-acetyl group of the α-APA inhibitor.

Thus, any NNI binding pocket model based on an individual RT-NNI crystal structure would have limited potential for predicting the binding of new, chemically distinct inhibitors. To overcome this problem, the invention disclosed herein uses the NNI binding site coordinates of multiple, varied RT-NNI structures to generate a composite molecular surface. A specific embodiment of the invention is a composite molecular surface or binding pocket generated from nine distinct RT-NNI complexes, and reveals a larger than presumed NNI binding pocket not shown or predicted by any of the individual structures alone (FIG. 2A). This novel composite binding pocket, together with a computer docking procedure and a structure-based semi-empirical score function, provides a guide to predict the energetically favorable position of novel PETT, DABO, and HEPT derivatives, as well as other novel compounds, in the NNI binding site of RT.

The invention further provides a number of computational tools which set forth a cogent explanation for the previously unexplained and not understood relative activity differences among NNIs, including PETT, DABO, and HEPT derivatives, and reveals several potential ligand derivatization sites for generating new active derivatives. Disclosed herein is the structure-based design of novel HEPT derivatives and the design and testing of non-cytotoxic PETT and DABO derivatives which abrogate HIV replication in human peripheral blood mononuclear cells at nanomolar concentrations with an unprecedented selectivity index of >10⁵.

One procedure useful in structure-based rational drug design is docking (reviewed in Blaney, J. M. and Dixon, J. S., Perspectives in Drug Discovery and Design, 1993, 1, 301). Docking provides a means for using computational tools and available structural data on macromolecules to obtain new information about binding sites and molecular interactions. Docking is the placement of a putative ligand in an appropriate configuration for interacting with a receptor. Docking can be accomplished by geometric matching of a ligand and its receptor, or by minimizing the energy of interaction. Geometric matching is faster and can be based on descriptors or on fragments.

Structure-based drug design efforts often encounter difficulties in obtaining the crystal structure of the target and predicting the binding modes for new compounds. The difficulties in translating the structural information gained from X-ray crystallography into a useful guide for drug synthesis calls for continued effort in the development of computational tools. While qualitative assessments of RT-inhibitor complexes provide helpful information, systematic quantitative prediction of inhibitory activity of new compounds based on structural information remains a challenge.

There is a need for more complete information on the structure and flexibility of the NNI binding pocket and for an improved model of the binding pocket to serve as a basis for rational drug design. In addition, there is a need for more effective inhibitors of reverse transcriptase, particularly HIV-1 reverse transcriptase.

The invention disclosed herein addresses these needs by providing a model for the three-dimensional structure of the RT-NNI binding pocket based on the available backbone structure of RT-DNA complex and full structure of RT complexed with several NNI compounds. Structural information from multiple RT-NNI complexes was combined to provide a suitable working model. In one embodiment, the NNI binding site coordinates of nine RT-NNI structures is used to generate a composite molecular surface revealing a larger than presumed NNI binding pocket. This pocket, together with docking and a structure-based semi-empirical score function, can be used as a guide for the synthesis and analyses of structure-activity relationships for new NNI of RT, including new derivatives of HEPT, DABO, and PETT, as well as novel compounds having little or no relationship to known NNIs. The practical utility of this novel composite model is illustrated and validated by the observed superior potency of new PETT and S-DABO derivatives as anti-HIV agents, described herein.

SUMMARY OF THE INVENTION

The invention provides novel compounds which inhibit reverse transcriptase (RT) and which inhibit replication of a retrovirus, such as human immunodeficiency virus-1 (HIV-1). In one embodiment, the novel compounds of the invention are analogs or derivatives of phenethylthiazolylthiourea (PETT), dihydroalkoxybenzyloxopyrimidine (DABO) or 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). Alternatively, the novel compounds of ID the invention bind the NNI binding pocket, but are not related to any known NNI. Specific compounds of the invention are described more fully in the Detailed Description and in the Examples below.

The invention additionally provides compositions and methods for inhibiting reverse transcriptase activity of a retrovirus, such as HIV-1, by contacting the RT binding site of the retrovirus with a compound of the invention. The methods of the invention are useful for inhibiting replication of a retrovirus, such as HIV-1 and include treating a retroviral infection in a subject, such as an HIV-1 infection, by administering a compound or composition of the invention, for example, in a pharmaceutical composition.

The invention further provides a composite ligand binding pocket constructed by superimposing multiple structures of ligand-binding site complexes. Preferably, the composite binding pocket is constructed by superimposing the structures of at least one each of the following NNI complexed with RT: a compound, analog or derivative of HEPT or MKC; TNK, APA, Nevipapine, and TIBO. In one embodiment, the composite ligand binding pocket is an HIV-1 reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket constructed by superimposing nine structures of NNI-RT complexes, preferably having the coordinates set forth in Table 9.

Using the novel composite binding pocket of the invention, compounds that bind to the NNI binding site of reverse transcriptase can be identified and/or screened. For example, a useful inhibitor is identified by analyzing the fit of a candidate compound to the composite binding pocket is analyzed. In one embodiment, the comparing comprises analyzing the molecular surface of the composite binding pocket. The extent of contact between the molecular surface of the compound and the molecular surface of the binding pocket can be visualized, and any gap space between the compound and the composite binding pocket can be determined and quantified. The candidate inhibitory compound can be docked in the composite binding pocket, and its binding characteristics analyzed. For example, an estimate of the inhibition constant for the docked compound can be calculated. The value of the inhibition constant is inversely related to the affinity of the candidate compound for the binding pocket.

Using information provided by the composite binding pocket of the invention, novel inhibitors of reverse transcriptase can be designed and screened. Using molecular modeling techniques, a compound can be docked into an RT-NNI binding pocket, and the complex analyzed for its binding characteristics. Gap space or regions that do not demonstrate optimum close contacts between the compound and the binding pocket are identified, permitting the compound to be modified to better occupy the site. In such a method, novel inhibitors of reverse transcriptase are designed and screened.

Also provided by the invention are inhibitors of reverse transcriptase identified or designed by analyzing the compound's structural fit to the binding pocket. Potent inhibitors designed and confirmed using the composite binding pocket of the invention include analogs and derivatives of known NNI, such as phenethylthiazolylthiourea (PETT) analogs, dihydroalkoxybenzyloxopyrimidine (DABO) analogs, and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs.

The compounds of the invention may be combined with carriers and/or agents to enhance delivery to sites of viral infection, such as targeting antibodies, cytokines, or ligands. The compounds may include chemical modifications to enhance entry into cells, or may be encapsulated in various known delivery systems.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a model of the HIV-1 reverse transcriptase (RT) active site, derived primarily from two crystal structures: HIV-1 RT (PDB access code hni) and HIV-1 RT with DNA fragment (PDB access code hmi). The binding site for non-nucleoside inhibitors is labeled NNI. The site for nucleoside inhibitors is labeled dNTP which includes the 3′ terminus of DNA. Features describing the geometry of the binding region include the thumb, palm, fingers, and hinge region of RT.

FIG. 1B shows models of compound I-3 (color coded by atom type) and compound I-4 (in blue) in NNI binding site of HIV reverse transcriptase, positioned by docking procedure. Wing 1 and Wing 2 represent two different regions of the NNI binding site.

FIG. 2A shows a composite binding pocket of NNI active site of HIV-1 RT. Grid lines represent the collective van der Waals surface of nine different inhibitor crystal structures superimposed in the active site and highlight the available space for binding (inhibitor structures include HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and two 9-Cl TIBO compounds, with PDB access codes rti, rt1, rt2, hni, vrt, rth, hnv, rev and tvr, respectively). The surface is color-coded for hydrogen bonding (red), hydrophobic (gray) and hydrophilic (blue) groups of the superimposed inhibitors. The hydrogen atoms were not included.

FIG. 2B shows a composite binding pocket (purple) superimposed on the active site residues of RT taken from the crystal structure coordinates of RT complexed with 8-Cl-TIBO(pdb access code: hnv). In the composite binding pocket, there are a number of regions which are larger than those defined by residues in individual crystal structures. Residues shown here which extend past the purple surface and toward the center of the binding site represent regions which are considered flexible and could be displaced by an appropriate inhibitor.

FIG. 3A shows a model of compound trouvirdine docked in the NNI binding site and color-coded by atom type. Spheres represent the sites of the molecular surface which are in contact with protein residues and are unavailable for future modification.

FIG. 3B shows a model of PETT compound I-3 docked in the NNI binding site and color-coded by atom type. Spheres represent the sites of the molecular surface which are in contact with protein residues and are unavailable for future modification.

FIG. 4A shows a stereo model of compound I-2 and grid shown in red which represents gaps between the compound and protein residues (each red line=1 Å distance). Dashed lines show the nearest distance between an atom in the compound and the gap net which does not intersect the spheres shown in FIG. 3A.

FIG. 4B shows a stereo model of PETT compound I-3 and grid shown in red which represents gaps between the compound and protein residues (each red line=1 Å distance). Dashed lines show the nearest distance between an atom in the compound and the gap net which does not intersect the spheres shown in FIG. 3B.

FIG. 5A shows a stereoview of compound trovirdine in the composite binding pocket which was constructed from combined coordinates of RT complexed with nine different NNI compounds.

FIG. 5B shows a stereoview of PETT compounds I-3 (in magenta) and I-4 (multicolor) in the composite binding pocket which was constructed from combined coordinates of RT complexed with nine different NNI compounds.

FIG. 6 shows a model of PETT compound II-4 docked in the NNI binding site and color-coded by atom type, as described above for FIG. 3A. The surface of the composite binding pocket is color-coded for hydrogen bonding (red), hydrophobic (gray) and hydrophilic (blue) groups of the superimposed inhibitors.

FIG. 7A is a view of the composite binding pocket of the NNI active site of HIV-1 RT. The DABO compound 3c is superimposed in the NNI composite binding site of the crystal structure of the RT/MKC-442 complex (hydrogen atoms not shown). MKC-442 (from crystal structure) is shown in pink, and compound 3c (from docking calculations) in multicolor. Compound 3c was docked into the active site of the RT/MKC complex (PDB access code: rt1) and then superimposed into the NNI composite binding pocket based on the matrix used in the pocket construction. The S2 substituent of the DABO compound 3c occupies the same region of the binding pocket as the N1 substituent of the HEPT derivative MKC-442.

FIG. 7B is a view of the composite binding pocket of the NNI active site of HIV-1 RT. An X-ray crystal structure of DABO compound 3b is superimposed on the docked model of DABO compound 3d in the NNI composite binding pocket of RT, demonstrating their remarkably similar conformations.

FIG. 8 is an ORTEP drawing of the room temperature X-ray crystal structure of DABO compound 3b (30% ellipsoids).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

As used herein, a “retrovirus” includes any virus that expresses reverse transcriptase. Examples of a retrovirus include, but are not limited to, HIV-1, HIV-2, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV, and MoMuLV.

As used herein, “reverse transcriptase (RT)” refers to an enzyme having an NNI binding site similar to that of HIV-1 RT and to which ligands which bind the composite binding pocket of the invention bind.

As used herein, “reverse transcriptase (RT) activity” means the ability to effect reverse transcription of retroviral RNA to proviral DNA. One means by which RT activity can be determined is by measuring viral replication. One measure of HIV-1 viral replication is the p24 core antigen enzyme immunoassay, for example, using the assay commercially available from Coulter Corporation/Immunotech, Inc. (Westbrooke, Mich.). Another means by which RT activity is analyzed is by assay of recombinant HIV-1 reverse transcriptase (rRT) activity, for example, using the Quan-T-RT assay system commercially available from Amersham (Arlington Heights, Ill.) and described in Bosworth, et al., Nature 1989, 341:167-168.

As used herein, a compound that “inhibits replication of human immunodeficiency virus (HIV)” means a compound that, when contacted with HIV-1, for example, via HIV-infected cells, effects a reduction in the amount of HIV-1 as compared with untreated control. Inhibition of replication of HIV-1 can be measured by various means known in the art, for example, the p24 assay disclosed herein.

As used herein, a “nonnucleoside inhibitor (NNI)” of HIV reverse-transcriptase (HIV-RT) means a compound which binds to an allosteric site of HIV-RT, leading to noncompetitive inhibition of HIV-RT activity. Examples of nonnucleoside inhibitors of HIV-RT include, but are not limited to, tetrahydroimidazobenzodiazepinthiones (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymines (HEPT), bis(heteroaryl)piperazines (BHAP), 2′-5′-bis-O-(tertbutyldimethylsilyl)-3′-spiro-5″-(4″-amino-1″,2″-oxathiole-2″,2″-dioxide)pyrimidines (TSAO), dihydroalkoxybenzyloxopyrimidine (DABO) and phenethylthiazolylthiourea (PETT) analogs. In one embodiment of the invention, the nonnucleoside inhibitor of HIV-RT is a PETT analog. In another embodiment of the invention, the nonnucleoside inhibitor of HIV-RT is a DABO analog. In another embodiment of the invention, the nonnucleoside inhibitor of HIV-RT is a HEPT analog.

As used herein, a “composite HIV reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket” or “composite binding pocket” means a model of the three-dimensional structure of a ligand binding site, such as the nonnucleoside inhibitor binding site of HIV-RT constructed from a composite of multiple ligand-binding site complexes. The composite binding pocket represents a composite molecular surface which reveals regions of flexibility within the binding site. Flexible residues within the NNI binding site include Tyr188, Tyr181, Tyr318, Tyr319, Phe227, Leu234, Trp229, Pro95, and Glu138 (the latter from the p51 subunit of RT) (SEQ ID NO:1). Examples of such a model include, but are not limited to, a composite molecular surface developed with the aid of computer software and based on a composite of coordinates of multiple RT-NNI complexes, as disclosed herein. In one embodiment, the binding pocket has the coordinates set forth in Table 9.

As used herein, a “compound that fits the nonnucleoside inhibitor (NNI) pocket of reverse transcriptase (RT)” means a compound that substantially enters and binds the NNI binding site on RT. In one embodiment, a compound that fits the NNI pocket of RT inhibits RT activity. Generally, compounds which better fit the NNI pocket of RT contact a greater portion of the available molecular surface of the pocket and are more potent inhibitors of RT activity. In one embodiment, the compound that fits the NNI pocket of RT is a PETT analog. In another embodiment, the compound that fits the NNI pocket of RT is a DABO analog. In another embodiment, the compound that fits the NNI pocket of RT is a HEPT analog.

As used herein, “docking” a compound in a binding pocket means positioning a model of a compound in a model of the binding pocket. In one embodiment, the model of the binding pocket can be a composite binding pocket constructed in accordance with the invention. The model of the binding pocket can be, for example, based on coordinates obtained from the crystal structure of RT complexed with a NNI. In one embodiment, the docking is performed with the use of computer software, such as the Affinity program within InsightII (Molecular Simulations Inc., 1996, San Diego, Calif.). Docking permits the identification of positions of the compound within the binding pocket that are favored, for example, due to minimization of energy.

As used herein, “minimization of energy” means achieving an atomic geometry of a molecule or molecular complex via systematic alteration such that any further minor perturbation of the atomic geometry would cause the total energy of the system as measured by a molecular mechanics force-field to increase. Minimization and molecular mechanics force-fields are well understood in computational chemistry (Burkert, U. and Allinger, N. L., Molecular Mechanics, ACS Monograph, 1982, 177, 59-78, American Chemical Society, Washington, D.C.).

As used herein, “comparing” includes visualizing or calculating available space encompassed by the molecular surface of the composite binding pocket of the invention, taking into account the flexibility of residues, such as Tyr188, Tyr181, Tyr318, Tyr319, Phe227, Leu234, Trp229, Pro95, and Glu138 of RT (the latter from the p51 subunit of RT) (SEQ ID NO:1). “Comparing” also includes calculating minimal energy conformations.

As used herein, “gap space” means unoccupied space between the van der Waals surface of a compound positioned within the binding pocket and the surface of the binding pocket defined by residues in the binding site. This gap space between atoms represents volume that could be occupied by new functional groups on a modified version of the compound positioned within the binding pocket.

In the present invention, the terms “analog” or “derivative” are used interchangeably to mean a chemical substance that is related structurally and functionally to another substance. An analog or derivative contains a modified structure from the other substance, and maintains the function of the other substance, in this instance, maintaining the ability to interact with an NNI-RT binding site. The analog or derivative need not, but can be synthesized from the other substance. For example, a HEPT analog means a compound structurally related to HEPT, but not necessarily made from HEPT.

As used herein, “alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.

As used herein, ÒalkeneÓ includes both branched and straight-chain unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms.

As used herein, “halogen” includes fluoro, chloro, bromo and iodo.

As used herein “non-hydrogen atom group” includes, but is not limited to, alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, thiol, thiolalkyl, amino, substituted amino, phosphino, substituted phosphino, or nitro. In addition, cycloalkyl, aryl, and aralkyl groups may be included if the non-hydrogen atom group contains a sufficient number of non-hydrogen atoms. Often, a number or range of numbers is specified to indicate the number of non-hydrogen (e.g., C, O, N, S, or P) atoms in the functional group.

As used herein, “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include, but are not limited to, (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or (c) salts formed with an organic cation formed from N,N′-dibenzylethylenediamine or ethylenediamine; or (d) combinations of (a) and (b) or (c), e.g., a zinc tannate salt; and the like. The preferred acid addition salts are the trifluoroacetate salt and the acetate salt.

As used herein, “pharmaceutically acceptable carrier” includes any material which, when combined with a compound of the invention, allows the compound to retain biological activity, such as the ability to inhibit RT activity, and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.

Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Col, Easton Pa. 18042, USA).

Composite Binding Pocket of the Invention

As shown in FIG. 1, the NNI binding site of HIV-RT rests between the palm and thumb regions of the RT molecular structure, adjacent to the hinge region. The NNI binding site includes two distinct regions, indicated in FIG. 1B as Wing 1 and Wing 2, forming a butterfly-shaped binding pocket.

In the method of the invention, a composite ligand binding pocket is constructed by superimposing multiple structures of ligand-binding site complexes, preferably using 5 or more distinct structures. In one embodiment, the composite ligand binding pocket is an HIV-1 reverse-transcriptase (RT) nonnucleoside inhibitor (NNI) binding pocket constructed by superimposing multiple structures of NNI-RT complexes. The composite binding pocket is preferably an HIV-1 RT-NNI binding pocket.

A preferred binding pocket of the invention can be made by superimposition of coordinates, obtainable from the Protein Data Bank (PDB) via access codes disclosed herein, corresponding to the three-dimensional structure of an RT-NNI complex. The superimposition of coordinates is preferably based on alignment of the coordinates corresponding to the palm region of the binding pocket due to the greater rigidity of this region.

The superimposing of coordinates can also be accomplished by first using models of the protein backbone and DNA phosphate backbone of the RT-DNA complex structure (with PDB access code hmi) onto a model of an RT mutant complexed with an NNI, such as APA ((2-acetyl-5-methylanilino)(2,6-dibromophyl)acetamide) having PDB access code hni. Next, models of one or more additional RT-NNI complexes are superimposed onto the models superimposed above. In one embodiment, the superimposition is based on alignment of the region of RT from residue 100 to 230 (SEQ ID NO:1), preferably by a least squares procedure. In another embodiment, the superimposition is based on alignment of the region of RT from residues 97 to 213 (SEQ ID NO:1). Preferably, the superimposition is based on alignment of the palm region and part of the NNI binding site. Most preferably, the superimposition is based on alignment of the region corresponding to residues 100 to 230 of RT (SEQ ID NO:1), or on alignment of 117 C alpha atoms of residues 97 to 213 (SEQ ID NO:1), and preferably using a least squares procedure.

A molecular surface of a binding pocket can then be generated that encompasses all superimposed NNI models. One such composite binding pocket constructed from nine individual NNI-RT complex structures, is shown in FIG. 2A. Grid lines in the figure represent the collective van der Waals surface, and highlight space available for binding.

The molecular surface of the complex can be generated, for example, by reading the overlaid coordinates of the complexed inhibitors into a computer program such as GRASP (A. Nicholls, GRASP, Graphical Representation and Analysis of Surface Properties, 1992, New York). Examples of NNI compounds which can be used in the construction of a binding pocket include, but are not limited to, HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and 9-Cl TIBO (PDB access codes, rti, rt1, rt2, hni, vrt, rth, hnv and rev or tvr, respectively).

Using the composite NNI binding pocket, binding of compounds can be modeled to identify available space within the binding pocket. New and more potent NNI inhibitors of RT can be developed by designing compounds to better fit the binding pocket.

In one embodiment, the composite binding pocket is constructed by superimposing structures of NNI-RT complexes comprising RT complexed with: an HEPT or MKC analog; a TNK analog; an APA analog; a Nevirapine analog; and a TIBO analog. In another embodiment, the composite NNI binding pocket is based on the structure of RT complexed with 9 NNI and on the RT-DNA complex. Examples of NNI compounds which can be used in the construction of a binding pocket include, but are not limited to, HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 8-Cl TIBO, and 9-Cl TIBO structures (PDB access codes, rti, rt1, rt2, hni, vrt, rth, hnv, and tvr and/or rev, respectively). In one embodiment, the resulting composite binding pocket has the coordinates set forth in Table 9.

Construction and use of the Binding Pocket

A compound that binds the NNI binding site of reverse transcriptase is identified by comparing a test compound to the composite binding pocket of the invention and determining if the compound fits the binding pocket. As shown in FIGS. 7A and 7B, the test compound may be compared to another inhibitory compound, by superimposing the structures in the binding pocket. The test compound is also compared to the binding pocket by calculating the molecular surface of the compound complexed with the composite binding pocket. The extent of contact between the molecular surface of the compound and the molecular surface of the binding pocket can be visualized, and the gap space between the compound and the binding pocket can be calculated. In FIGS. 4A and 4B, gaps between the molecular surface of the binding pocket and the NNI are presented in red, with each red line being 1 angstrom in distance.

To design a novel inhibitor of reverse transcriptase, a compound is docked in the composite binding pocket of the invention. Gap space is identified between the compound and the binding pocket, for example, using an algorithm based on a series of cubic grids surrounding the docked compound, with a user-defined grid spacing. The compound is then modified to more completely occupy the gap space.

Computerized docking procedures can be used to dock the test compound in the binding pocket and analyze the fit. One docking program, DOCK (Kuntz, I. D., et al., J. Mol. Biol., 1982, 161, 269-288; available from University of California, San Francisco), is based on a description of the negative image of a space-filling representation of the receptor that should be filled by the ligand. DOCK includes a force-field for energy evaluation, limited conformational flexibility and consideration of hydrophobicity in the energy evaluation. CAVEAT (Bartlett, P. A. et al., Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc., 1989, 78, 182-196; available from University of California, Berkeley) suggests ligands to a particular receptor based on desired bond vectors. HOOK (Molecular Simulations, Burlington, Mass.) proposes docking sites by using multiple copies of functional groups in simultaneous searches. MACCS-3D (Martin, Y. C., J. Med. Chem., 1992, 35, 2145-2154) is a 3D database system available from MDL Information Systems, San Leandro, Calif. Modeling or docking may be followed by energy minimization with standard molecular mechanics forcefields or dynamics with programs such as CHARMM (Brooks, B. R. et al., J. Comp. Chem., 1983, 4, 187-217) or AMBER (Weiner, S. J. et al., J. Am. Chem. Soc., 1984, 106, 765-784).

LUDI (Bohm, H. J., J. Comp. Aid. Molec. Design, 1992, 6, 61-78; available from Biosym Technologies, San Diego, Calif.) is a program based on fragments rather than on descriptors. LUDI proposes somewhat larger fragments to match with the interaction sites of a macromolecule and scores its hits based on geometric criteria taken from the Cambridge Structural Database (CSD), the Protein Data Bank (PDB) and on criteria based on binding data. Other software which can be used to propose modifications for constructing novel inhibitors include LEGEND (Nishibata, Y. and Itai, A., Tetrahedron, 1991, 47, 8985; available from Molecular Simulations, Burlington, Mass.) and LeapFrog (Tripos Associates, St. Louis, Mo.).

The AUTODOCK program (Goodsell, D. S. and Olson, A. J., Proteins: Struct. Funct. Genet., 1990, 8, 195-202; available from Scripps Research Institute, La Jolla, Calif.) helps in docking ligands to their receptive proteins in a flexible manner using a Monte Carlo simulated annealing approach. The procedure enables a search without bias introduced by the researcher. This bias can influence orientation and conformation of a ligand in the active site. The starting conformation in a rigid docking is normally biased towards an energy minimum conformation of the ligand. However, the binding conformation of the ligand may be of relatively high conformational energy, but offset by the binding energy.

In a preferred embodiment of the invention, docking is performed by using the Affinity program within InsightII (Molecular Simulations Inc., 1996, San Diego, Calif.). As modeling calculations progress during the docking procedure, residues within a defined radius of 5 Å from the NNI molecule are allowed to move in accordance with energy minimization, permitting the identification of promising positions for modification. Initial coordinates of newly designed compounds can be generated using the Sketcher module within InsightII.

In one embodiment, the method further comprises calculating the inhibition constant of the docked compound. Inhibition constants (K_(i) values) of compounds in the final docking positions can be evaluated using a score function in the program, LUDI (Bohm, H. J., J. Comput. Aided Mol. Des., 1994, 8, 243-256; Bohm, H. J., J. Comput. Aided Mol. Des., 1992, 6, 593-606). Predictions of K_(i) values can be improved by modifications of the LUDI calculation., for example, those described in Example 1. First, the molecular surface area can be directly calculated from the coordinates of the compounds in docked conformation using the MS program described in Connolly, M. L., 1983 Science 221:709-713. Second, because InsightII does not account for structural rigidity imposed by internal hydrogen bonds, the number of rotatable bonds can be re-evaluated. For example, this re-evaluation can be performed by counting the number of rotatable bonds according to the principle introduced by Bohm (supra) and taking out the number of bonds which are not rotatable due to the conformational restraint imposed by the internal hydrogen bond between the thiourea NH and pyridyl N in PETT derivatives. Third, the calculation can be modified by the assumption that the conserved hydrogen bond with RT does not deviate significantly from the ideal geometry. This assumption is supported by the fact that, in known crystal structures of RT complexes, all hydrogen bonds between NNIs and RT are near the ideal geometry. These constraints provide for more predictive K_(i) values for modeled compounds.

In a preferred embodiment, the compound has a predicted inhibition constant (K_(i)) of less than about 1 μM, and the compound in the binding has an estimated molecular surface area greater than 276 Å².

Candidate inhibitors of RT identified or designed by the methods of the invention can be evaluated for their inhibitory activity using conventional techniques which typically involve determining the location and binding proximity of a given moiety, the occupied space of a bound inhibitor, the deformation energy of binding of a given compound and electrostatic interaction energies. Examples of conventional techniques useful in the above evaluations include, but are not limited to, quantum mechanics, molecular dynamics, Monte Carlo sampling, systematic searches and distance geometry methods (Marshall, G. R., Ann. Ref. Pharmacol. Toxicol., 1987, 27, 193). Examples of computer programs for such uses include, but are not limited to, Gaussian 92, revision E2 (Gaussian, Inc. Pittsburgh, Pa.), AMBER version 4.0 (University of California, San Francisco), QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass.), and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif.). These programs may be implemented, for example, using a Silicon Graphics Indigo2 workstation or IBM RISC/6000 workstation model 550. Other hardware systems and software packages will be known and of evident applicability to those skilled in the art.

Inhibitors identified or designed by the methods of the invention can be tested for their anti-HIV or anti-RT activity using one of the standard in vitro assays known in the art, such as the p24 enzyme immunoassay disclosed herein.

The invention further provides novel compounds identified by the above methods, which can be used as inhibitors of RT. Novel inhibitors so identified include analogs or derivatives of known NNI compounds such as HEPT, DABO, and PETT, as well as novel compounds designed to fit the composite binding pocket which are unrelated to any known NNI compound.

Compounds of the Invention

Compounds of the invention are useful as nonnucleoside inhibitors of RT. These include, for example, analogs and derivatives of PETT, DABO, and HEPT compounds, as well as novel compounds unrelated to known NNI but designed to fit the composite binding pocket.

PETT Compounds

Novel compounds of the invention include derivatives and analogs of PETT, having the general formula (I):

Z can be phenyl, piperizine, piperidine, or morpholine. Z is preferably substituted with one or more substituents, including alkyl, alkene, halogen, methoxy, alcohol, amino, thio, thioxy, or phosphino. In one embodiment, the compounds of the invention are PETT derivatives or analogs having the following formula (II):

The R's can be the same or different, and represent points of optional substitution. R₂, R₃, R₄, R₅, R₆, R₇ and R₈ can be hydrogen, or can be substituted, with a non-hydrogen atom group such as halo (Br, Cl, F, I), alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH₂ group, where R is alkyl. Preferably, one or more is alkyl, halo, or alkoxy. Preferred halogens are F, Br, or Cl. One or more of R₂, R₃, R₄, R₅, R₆, and R₇ can be a C1-C3 alkoxy, e.g., methoxy.

R₈ can also be aryl, aralkyl, ROH, or RNH₂ group, where R is alkyl. Preferably, at least one of R₂, R₃, R₄, R₅, and R₆ is not hydrogen. R₄ is a preferably hydrophobic group such as H, an alkyl or alkene, and can be Me, Et, or i-Pr. R₆ and/or R₇ are preferably a 3 or 4 (non-hydrogen)-atom group.

R₆ and R₇ can be a group having 1 to 4 non-hydrogen atoms, whereas R₂, R₃, and R₅ preferably each are a group having 1 to 3 non-hydrogen atoms. Available gap space in the binding pocket near R₈, is approximately 8 angstroms by 5 angstroms, by 3.3 angstroms. Thus, a molecule having a volume of up to about 8×6×4 angstroms can be used to fill this space, e.g., accommodating a group of about 7 non-hydrogen atoms, or up to about the size of a phenyl ring. R₈ can be halo, alkyl, phenyl, —CH₂Ph, or alkoxy. R₈ can be X—R, where X is a bridging atom, including, but not limited to, C, S, O, N and P.

In a preferred embodiment, R₈ is bromine, and at least one of R₂, R₃, R₄, R₅, and R₆ is fluoro, chloro, or methoxy.

A compound of the invention preferably conforms to the composite NNI binding pocket of the invention. Most preferably, the compound complexed with an NNI-RT binding pocket, has a predicted K_(i) of less than about 1 μM.

Preferred modifications of PETT compounds include ortho-halogen, meta-O-Me, and hydrophobic groups at the para position of the ring. Most preferably, the modifications do not disrupt the intramolecular hydrogen bond. Specific compounds include those having the following formulae (III-VIII) shown below.

In another embodiment, the PETT derivative comprises the formula (IX):

The R's can be the same or different, and represent points of optional substitution. R₅, R₆, and R₇ can be hydrogen, or can be substituted, with a non-hydrogen atom group such as halo (Br, Cl, F, I), alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH₂ group, where R is alkyl. Preferably, one or more is alkyl, halo, or alkoxy. Preferred halogens are F, Br, or Cl. One or more of R₅, R₆, and R₇ can be a C1-C3 alkoxy, e.g., methoxy.

R₆ and/or R₇ are preferably a 3 or 4 (non-hydrogen)-atom group. R₆ and R₇ can be a group having 1 to 4 non-hydrogen atoms, whereas R₅ preferably is a group having 1 to 3 non-hydrogen atoms. R₈ can be a group of about 7 non-hydrogen atoms, or up to about the size of a phenyl ring. R₈ can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, aryl, aralkyl, —CH₂Ph, alkoxy, ROH or RNH₂, where R is alkyl. R₈ can be X—R, where X is a bridging atom, including, but not limited to, C, S, O, N and P.

X can be CR′R″, NR′″, or O, where R′, R″, and R′″ can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, or phosphino group. In one embodiment, R₅, R₆, R′, R″, and R′″ are each hydrogen. In an alternative embodiment, X is CR′R″ and at least one of R′ and R″ are fluoro, chloro, bromo, hydroxy, methoxy, or C1-3 alkyl. In a preferred embodiment, R₈ is bromine, and at least one of R₅, R₆, and R₇ is fluoro, chloro, or methoxy.

Preferred compounds include a larger functional group near the ethyl linker, for example R₇ acetamide or methoxy. Also preferred is a bulkier heterocyclic ring such as a bulky piperidinyl ring or an ortho/meta substituted pyridyl ring.

Specific PETT derivatives of the invention include:

N-[2-(1-piperidinoethyl)]-N′-[2-(5-bromopyridyl)]thiourea,

N-[2-(2,5-dimethoxyphenethyl)]-N′-[2-(5-bromopyridyl)]thiourea,

N-[2-(o-Chlorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea

N-[2-(o-Fluorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea, and

N-[2-(m-Fluorophenethyl)]-N′-[2-(5-bromopyridyl)]thiourea.

Other specific compounds of the invention are described in the Examples below.

DABO Compounds

In another embodiment of the invention, the compounds are derivatives of DABO, and have the following general formula (X):

R₁ and R₂ can be alike or different, and can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. Preferably, one or more of R₁ and R₂ is a C1-3 alkyl, such as methyl (Me), ethyl (Et), or isopropyl (i-Pr). Preferably, R₁ is alkyl, alkenyl, ROH, or RNH₂. R₂ is preferably halo, alkyl, or C1-3 alkoxy.

Y can be S or O, and is preferably S. R3 can be alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl, and is preferably C1-3 alkyl.

Specific DABO compounds of the invention include:

5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one.

Other specific compounds of the invention are described in the Examples below.

HEPT Compounds

In another embodiment, the compounds of the invention are HEPT derivatives having the formula (XI):

X and Y can be independently S or O. Preferably, at least one of X and Y is S. More preferably, X is S, and in specific embodiments, both X and Y are S.

R₁ and R₂ can be hydrogen, halo, alkyl, alkenyl, hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is alkyl. R₃ can be H, alkyl, alkenyl, aryl, aralkyl, ROH, or RNH group, where R is alkyl. Preferably, R₁ is alkyl, alkenyl, ROH, or RNH₂, and can be, for example, methyl, ethyl, or isopropyl. R₂ is preferably halo, alkyl, or C1-3 alkoxy, and is preferably in the ortho or meta position. R₂ can be Br, F, Cl, or O—Me.

Specific HEPT compounds of the invention include:

6-benzyl-5-isopropyl-1[(methylthio)methyl]-2-thiouracil.

Other specific compounds of the invention are described in the Examples below.

The compounds of the invention have the ability to inhibit replication of a retrovirus, such as human immunodeficiency virus (HIV), preferably with an IC₅₀ of less than 50 μM, for example, as determined by p24 enzyme immunoassay described in the Examples below. More preferably, the compound of the invention inhibits replication of HIV in the p24 assay with an IC₅₀ of 1 to 5 μM, or less. Most preferably, the compound inhibits replication of HIV in the p24 assay with an IC₅₀ of less than 5 nM. In some embodiments, the compound inhibits replication of HIV in the p24 assay with an IC₅₀ of less than 1 nM.

The invention provides a composition comprising a compound or inhibitor of the invention, and optionally, an acceptable carrier. The composition can be a pharmaceutical composition. Compositions of the invention are useful for prevention and treatment of retroviral infection, such as HIV infection.

Methods of Using Compounds of the Invention

The compounds of the invention are useful in methods for inhibiting reverse transcriptase activity of a retrovirus. Retroviral reverse transcriptase is inhibited by contacting RT in vitro or in vivo, with an effective inhibitory amount of a compound of the invention. The compounds of the invention also inhibit replication of retrovirus, particularly of HIV, such as HIV-1. Viral replication is inhibited, for example, by contacting the virus with an effective inhibitory amount of a compound of the invention.

Due to the ability to inhibit replication of retrovirus and to inhibit retroviral RT activity, the invention provides a method for treating or preventing retroviral infection, such as HIV infection, and a method for treating AIDS or AIDS-related complex (ARC). The method comprises administering to a subject an effective inhibitory amount of a compound of the invention or a pharmaceutically acceptable salt of the compound. The compound or inhibitor of the invention is preferably administered in combination with a pharmaceutically acceptable carrier, and may be combined with specific delivery agents, including targeting antibodies and/or cytokines. The compound or inhibitor of the invention may be administered in combination with other antiviral agents, immunomodulators, antibiotics or vaccines.

The compounds of the invention can be administered orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. Pharmaceutical compositions of the invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.

For oral administration as a suspension, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation. The compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents. As immediate release tablets, the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

For administration by inhalation or aerosol, the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation. The compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

For rectal administration as suppositories, the compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquify or dissolve in the rectal cavity to release the drug.

Dosage levels of approximately 0.02 to approximately 10.0 grams of a compound of the invention per day are useful in the treatment or prevention of retroviral infection, such as HIV infection, AIDS or ARC, with oral doses 2 to 5 times higher. For example, HIV infection can be treated by administration of from about 0.1 to about 100 milligrams of compound per kilogram of body weight from one to four times per day. In one embodiment, dosages of about 100 to about 400 milligrams of compound are administered orally every six hours to a subject. The specific dosage level and frequency for any particular subject will be varied and will depend upon a variety of factors, including the activity of the specific compound the metabolic stability and length of action of that compound, the age, body weight, general health, sex, and diet of the subject, mode of administration, rate of excretion, drug combination, and severity of the particular condition.

The compound of the invention can be administered in combination with other agents useful in the treatment of HIV infection, AIDS or ARC. For example, the compound of the invention can be administered in combination with effective amounts of an antiviral, immunomodulator, anti-infective, or vaccine. The compound of the invention can be administered prior to, during, or after a period of actual or potential exposure to retrovirus, such as HIV.

Strategies for Design and Synthesis of Inhibitors

It has been proposed that NNI interfere with reverse transcription by altering either the conformation or mobility of RT rather than directly preventing the template-primer binding (Tantillo, C. et al., J. Mol Biol, 1994, 243, 369-387). Specifically, binding of NNI to the NNI binding site (approximately 10 Å away from the polymerase catalytic site) inhibits RT by interfering with the mobility of the “thumb” and/or position of the “primer grip” (residues 229-231), which interact with the DNA primer strand (FIG. 1A).

Computer programs can be used to identify unoccupied (aqueous) space between the van der Waals surface of a compound and the surface defined by residues in the binding site. These gaps in atom-atom contact represent volume that could be occupied by new functional groups on a modified version of the lead compound. More efficient use of the unoccupied space in the binding site could lead to a stronger binding compound if the overall energy of such a change is favorable. A region of the binding pocket which has unoccupied volume large enough to accommodate the volume of a group equal to or larger than a covalently bonded carbon atom can be identified as a promising position for functional group substitution. Functional group substitution at this region can constitute substituting something other than a carbon atom, such as oxygen. If the volume is large enough to accommodate a group larger than a carbon atom, a different functional group which would have a high likelihood of interacting with protein residues in this region may be chosen. Features which contribute to interaction with protein residues and identification of promising substitutions include hydrophobicity, size, rigidity and polarity. The combination of docking, K_(i) estimation, and visual representation of sterically allowed room for improvement permits prediction of potent derivatives.

Design of HEPT Derivatives

New HEPT derivative designs included compounds with added groups at the N-1 (Y—R3) and C-5 (R₁) positions and those having oxygen (X or Y) atoms replaced by sulfur. Substitution of oxygen by sulfur can aid binding by decreasing the desolvation energy involved in binding. The modifications were made such that the HEPT derivative would fit favorably into the butterfly-shaped RT-NNI binding site, (See FIG. 2A) with the benzyl ring residing in one wing and thymine ring in the other. For all designed compounds, the benzyl ring is near Trp229 and the N-1 group is near Pro236, a typical position observed in crystal structures. The modeling calculations, along with the application of the constructed binding pocket, provided a guideline for the synthesis of lead compounds designed to have potent anti-HIV activity. The choice of compounds was also based on synthetic feasibility.

The region of the NNI site of HIV-1 RT located near the thymine ring nitrogen N-1 of the HEPT analogs contains a Pro236 loop region which is large enough to accommodate N-1 substituents. When an inhibitor binds to the NNI site of HIV-1 RT, the presence of a hydrophobic N-1 substituent could influence the Pro loop of this flexible region and provide additional hydrophobic contact leading to stronger binding. Docking results indicated that substitution at N-1 also helps the molecule position itself to achieve the best fit within the pocket.

The LUDI analysis showed a substantial increase in contact (lipo score) between the compound and the pocket and the calculation suggested an increase in hydrophobic contact and stronger binding when the substituent on the N-1 tail (R₃) is larger in size than a methyl moiety.

The Tyr183 residue of the HIV-1 RT is located in the catalytic region which has a conserved YMDD motif characteristic of reverse transcriptases. Therefore, the displacement of this tyrosine residue can interfere with catalysis and render the HIV-1 RT protein inactive. It has been suggested that bulky substituents at the 5th position of the thymine ring (R₁) could indirectly accomplish this goal by displacing Tyr181 which is near Tyr183. The composite binding pocket shows sufficient room for at least a 3-carbon group in this region. The addition of a methyl, ethyl or isopropyl group on the 5th position of the thymine ring would lead to a higher affinity for the relatively hydrophobic environment.

LUDI analysis showed that the hydrophobic contact increases as hydrophobic groups at the 5th position (R₁) get bulkier. As it binds to the site, the ethyl or isopropyl group causes the nearby Tyr181 residue to rotate away from the inhibitor. This change in conformation in turn affects the positions of the neighboring Tyr183 and Tyr188 which can lead to the inactivation of HIV-1 RT.

DABO Derivatives

Detailed analysis of HEPT binding revealed that the N1 substituents of HEPT derivatives occupy the same region of the binding site as the thio (S2) substituents of DABO compounds (See FIG. 7A). Therefore, new DABO derivatives were designed and their binding into the NNI site of RT modeled using the crystal structure coordinates of the RT/MKC complex (pdb access code: rt1) and a molecular docking procedure. The final coordinates of the docked molecules were then superimposed into the composite binding pocket to evaluate the fit within the RT NNI pocket. Notably, multiple sterically allowed unoccupied spatial gaps in the binding site were identified from the docking studies which could be filled by strategically designed functional groups (See FIG. 7B).

The docked DABO molecule showed significant space surrounding the 6-benzyl ring and the 5th position of the thymine ring, which led to our design and synthesis of new DABO derivatives. Specific DABO compounds are discussed more fully in the Examples, below.

Each PETT derivative described in the Examples below, can be viewed as two chemical groups linked together by a thiourea group. Upon binding RT, the PETT derivative fits into the butterfly-shaped binding site. (See FIG. 6). One half of the molecule is composed of a pyridyl thiourea group (compounds I-1 to 4, II-1 to 9, and III-1 to 3) or a 2-aminothiazole group (PETT) which forms an intramolecular hydrogen-bonded 6-membered heterocyclic ring (shown below). The other half of the molecule is a piperidinyl ring (II-9), a pyridyl ring (trovirdine), or a phenyl ring separated from the thiocarbonyl group by an ethyl linker.

The positions of the compounds having stronger binding and higher scores (evaluated by LUDI function) all fall into the butterfly-shaped binding region with one part residing in Wing 1 and the other in Wing 2, as illustrated in FIG. 1B. For these compounds the ring closest to the thiocarbonyl group is near the Lys(K)101 loop and the other pyridyl ring is near Trp(W)229 derivatives.

Analysis of trovirdine, revealed multiple sites which can be used for the incorporation of larger functional groups. In the composite binding pocket, the docked trovirdine molecule showed a lot of usable space surrounding the pyridyl ring, (R₂-R₆), the ethyl linker (R₇) and near the 5-bromo position (R₈). (See FIG. 5A)

Efficient use of this space by strategically designed functional groups would lead to high affinity binding and ultimately result in better inhibitors. Our modeling studies suggest that designs using the space available in these regions, including (1) substitutions at R₂-R₆; (2) substituting heterocyclic rings for the pyridyl ring of trovirdine; (3) substitutions at R₇; (4) substitutions at R₈; and (5) maintaining the intramolecular hydrogen bond. As shown in the Examples below, modifications in these areas lead to potent RT inhibitors.

Advantage of the Invention

The invention provides a model for the three-dimensional structure of the RT-DNA complex based on the available backbone structure of RT-DNA complex and full structure of RT complexed with several NNI compounds. This is the first model to combine structural information from several complexes into a single composite and provides a suitable working model for the development of novel inhibitory compounds. The use of multiple NNI binding site coordinates from RT-NNI structures, as disclosed herein, permits the generation of a composite molecular surface. Analysis of the composite NNI binding pocket of the invention reveals that the binding pocket is surprisingly and counter-intuitively larger (instead of smaller) and more flexible (instead of more rigid) than expected. This composite NNI binding pocket serves as a guide for the synthesis and analyses of structure-activity relationships for the identification and design of new and more potent NNI of RT. The composite binding pocket additionally provides a model for the design of derivatives of NNIs for which crystal structure information is not available (e.g., PETT, DABO).

The compounds of the invention are useful for inhibition of RT activity and for inhibition of retroviral replication. The compounds disclosed herein provide more potent NNI of RT than known HEPT, DABO and PETT derivatives. With all strategies combined, a number of sites are identified for developing more potent derivatives of PETT, such as the incorporation of a larger functional group near the ethyl linker of PETT. Hitherto unknown piperidinyl substituted and piperozinyl substituted, as well as morpholinyl substituted PETT derivatives are disclosed which show potent anti-HIV activity at nanomolar concentrations.

In addition, the compounds of the invention provide a higher selectivity index (S.I.>10⁵) than currently available anti-HIV compounds. This high S.I. permits more effective antiviral activity with a minimum of adverse cytotoxic effects.

EXAMPLES

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

Example 1 Modeling Procedure

Construction of the Composite NNI Binding Pocket

A novel model of the NNI binding pocket of RT was constructed by superimposing nine individual RT-NNI crystal structures and then generating a van der Waals surface which encompassed all of the overlaid ligands. This “composite binding pocket” surprisingly reveals a different and unexpectedly larger NNI binding site than shown in or predictable from any of the individual structures and serves as a probe to more accurately define the potentially usable space in the binding site (FIG. 2A).

Modeling studies were based on the construction of a binding pocket which encompassed the superimposed crystal structure coordinates of all known RT-NNI complexes, including nine different structures of RT complexed with HEPT, MKC, TNK, APA, Nevirapine, N-ethyl Nevirapine derivative, 9-Cl TIBO (Ren, J. et al., Structure, 1995, 3, 915-926); 9-Cl TIBO (Das, K. et al., J. Mol. Biol., 1996, 264, 1085-1100) and 8-Cl-TIBO (PDB access codes rti, rt1, rt2, hni, vrt, rth, rev, tvr, and hnv, respectively).

The “thumb” region of RT complexes are relatively variable compared with the “palm” region. Therefore, a total of 117 C-alpha atoms of the residues from 97 to 213 (SEQ ID NO:1) which cover part of the NNI binding site and the “palm” region were used for a least-squares superimposing procedure within the program O (Jones, T. A. et al., Acta Crystallogr. A., 1991, 47, 110-119). Each coordinate set was superimposed onto the same initial coordinate set (RT/9-Cl TIBO). The distance between the pair was minimized by rotating and translating one coordinate set onto the other, minimizing distances between x, y, and z coordinates, according to the method of the program “O”. The root mean square (RMS) values of the coordinates of the atoms being superimposed are shown to be 1.00, 0.98, 0.99, 0.62, 0.80, 0.87, 0.94 and 0.65 Å for HEPT, MKC, TNK, APA, Cyclopropanyl Nevirapine, N-ethyl Nevirapine derivative and two 9-Cl TIBO compounds, respectively. Next, the coordinates of the corresponding inhibitor molecules were then transformed according to the same matrices derived from the superimposition. Lastly, the overlaid coordinates of all inhibitors were read into the program GRASP (Nicholls, A., GRASP 1992, New York), from which an overall molecular surface was generated providing a binding pocket encompassing all inhibitors.

As shown in FIG. 2A, the surface of the binding pocket was color coded to reflect characteristics of the overlaid inhibitors, such as hydrogen bonding, hydrophilic, and hydrophobic regions. The amide nitrogens on the uracil ring of HEPT and TIBO derivatives are color-coded red for hydrogen bonding atoms. Oxygen or sulfur atoms of carbonyl, thiocarbonyl, and ester groups, nitrogen atoms of amine groups, and halogen atoms are color-coded blue for polar (hydrophilic) groups. Carbon atoms are considered hydrophobic and are colored grey. This pocket, referred to as the composite binding pocket, was used as a basis for the analysis of inhibitor binding.

To generate the coordinates of the composite binding pocket using the InsightII program, each data point of the net defining the surface of the pocket was represented as a water molecule and was saved in Brookhaven Protein Databank (pdb) format. To provide a visual frame of reference, the coordinates have been superimposed on the pdb coordinates of an existing crystal structure having pdb access code hnv (HIV-1 RT/8-Cl TIBO complex). The coordinates of a composite binding pocket for HIV-1 RT generated by superimposing nine different NNI-RT complexes, are set forth in Table 9.

Docking and K_(i) Prediction

A computer simulation of the binding of PETT, DABO, and HEPT compounds into the NNI binding site of RT was accomplished using a molecular docking procedure. Docking of the compounds into the NNI binding site required the use of X-ray coordinates of an RT-NNI complex (RT/9-Cl-TIBO complex was used for modeling PETT, and the RT/MKC-442 complex was used for modeling DABO and HEPT). Upon binding to RT, the compound can fit into a butterfly-shaped NNI binding site (described by Ding et. al), Ding, J. et al., Nat. Struct. Biol., 1995, 2, 407-415 (FIG. 1B and 2A). Once the final docked position of the molecule in the NNI site was determined, the molecule was assigned a score (LUDI), from which an estimation of the inhibition constant (K_(i) value) was determined.

After docking and K_(i) estimation was completed for the inhibitors, evaluation of the docked compounds in the active site of RT involved placing each compound into the composite binding pocket using the same orientation matrix utilized in construction of the pocket. The potentially flexible regions in the binding site were then readily identified as were atom sites for future derivatization of the compounds. Fixed docking in the Affinity program within InsightII (InsightII, Molecular Simulations Inc., 1996, San Diego, Calif.), was used for docking small molecules to the NNI binding site which was taken from a crystal structure (PDB code rev, RT/9-Cl-TIBO complex). The program has the ability to define a radius of residues within a 5 Å distance from the NNI molecule. As the modeling calculations progressed, the residues within the radius were allowed to move in accordance with the energy minimization. Ten final docking positions were initially chosen for each inhibitor modeling calculation but failed to reveal more than two promising positions. Later, only two calculated positions were set for the search target.

Calculations were carried out on a Silicon Graphics INIDIGO² using the CVFF force field in the Discover program and a Monte Carlo search strategy in Affinity (Luty, B. A. et al., J. Comp. Chem., 1995, 16, 454-464). No solvation procedures were used. Since the total number of movable atoms exceeds 200, Conjugated Gradient minimization was used instead of the Newton minimization method. The initial coordinates of the compounds were generated using the Sketcher module within InsightII. Each final docking position was then evaluated by a score function in LUDI. The top scoring model was then compared with the composite binding pocket and the known crystal structure of similar compounds and used for further analyses. The inhibitory constants (K_(i) values) of the positioned NNI compounds were evaluated using the LUDI score function (Bohm, H. J., J. Comput. Aided. Mol. Des., 1994, 8, 243-256; Bohm, H. J., J. Comput. Aided. Mol. Des., 1992, 6, 593-606).

Several modifications were imposed during the calculation of inhibitory constants (K_(i) values) of the positioned compounds using the LUDI score function (Bohm, H. J. 1994 supra; Bohm, H. J. 1992 supra). First, the molecular surface areas (MS) were directly calculated from the coordinates of the compounds in docked conformations using the MS program. Second, the number of rotatable bonds (NR), which was assessed inaccurately by INSIGHTII (rigidity imposed by hydrogen bonding was not accounted for in the program), was re-evaluated. Third, it was assumed that the conserved hydrogen bond with RT was assumed to not deviate significantly from the ideal geometry. This assumption was supported by the fact that in the known crystal structures of RT complexes, all hydrogen bonds between NNIs and RT are near the ideal geometry. Last, for the trovirdine compounds, an additional penalty was imposed for a charged group or halogen atoms when positioned near the ring plane of a protein residue such as tryptophan 229 because the interaction was not adequately accounted for in the LUDI score. The working modification of the LUDI scoring function for the PETT compounds included subtracting a score of P from the total LUDI score when the ring plane of the Trp229 was within 5 Å from a para substituent (R):

LUDI Score=MS*BS*2.93+85(H-bond)−NR*24.2−100−P;

where

P=200, when R=a hydrophilic group, e.g. —OH or —NO2;

P=100, when R=a para-halogen atom, e.g. —F, —Cl or —Br;

P=50, when R=a para-methoxy, e.g. —OMe;

P=0, when R=a hydrophobic group, e.g. H, CH3;

Consequently, the K_(i) values for the modeled compounds were more predictable than they would be without such modification (Bohm, H. J. 1994 supra; Bohm, H. J. 1992 supra).

Contact Surface and Gap Analysis

Independent of the composite binding pocket and as a follow-up to the docking procedure, computer programs were used to analyze the surface complementarity between the compounds and the binding site residues. This analysis provided another useful way to examine binding interactions, based solely upon the structure that was used for docking (RT/9-Cl TIBO for PETT and RT/MKC-442 for DABO and HEPT) (Das, K. et al., J. Mol. Biol., 1996, 264, 1085-1100).

A number of computer programs were written to analyze the surface of the compounds in the NNI binding site of RT and to better visualize any spatial gaps between the compounds and nearby residues of the RT protein. The algorithm used in these programs was based on a series of cubic grids surrounding the compound, with a user-defined grid spacing. All cubes were coded based on the distance and the nature of the interaction with the protein residues and/or compound atoms. The cubes that overlap both protein and compound within the contact radius are displayed as spheres and were selected to represent the buried surface (user-defined contact radius was the van der Waals radius plus an uncertainty factor, dependent on the reliability of source coordinates). All other cubes that did not interact with protein residues and were within a certain distance from the compound were selected to represent the gap space (space unoccupied by compound or protein) and are displayed as rods.

A graphic interface was then used to examine whether the “gap” spheres could be connected with the compounds without intersecting the “contact” spheres. If the criterion was met, the points that stemmed from the surface of the compound were defined as an expandable region (eligible for synthetic modification). The spheres generated by the programs (shown in FIG. 3) represent the sites buried by protein residues, indicating regions of the compound which are probably not available for derivatization.

FIG. 4 shows the binding pocket embellished with a grid of red rods which represent unoccupied space between the compound and active site residues, providing a complementary view to that shown by the spheres. The grid illustrates the candidate sites for derivatization of the compound and, when used as a distance scale (the length of one rod represents 1 Å), also indicates the volume available for new functional groups.

One example of a useful program is the “SeeGap” program, whose code is listed below in Example 11, together with instructions for its use.

Composite NNI Binding Pocket of RT Reveals Protein Flexibility and Future Inhibitor Modification Sites

The integrated structural information surprisingly revealed a much larger binding site than any shown in individual structures and served as a probe to define the potentially usable space in the binding site (FIG. 1). The three-dimensional binding site can be used as a reference point for the analysis of compounds which have been positioned by a docking procedure.

Upon inspection of the pocket it was apparent that although there are no large-scale conformational changes within the NNI binding site, a number of RT protein residues in contact with the inhibitors are relatively flexible and vary from structure to structure. These residues include Tyr188, Tyr181, Tyr318, Try319, Phe227, Leu234, Trp229, Pro95, and Glu138 latter from p51 subunit of RT).

As shown in FIG. 2B, the surface of the composite binding pocket which is overlaid with the RT-TIBO binding site is a short distance (<1.5 Å) away from or even extends past RT residues 234-236, Y188, F227, and the backbone of K101 (SEQ ID NO:1). This indicates that these residues are flexible and can be displaced by the right substituent on an inhibitor.

The composite binding pocket of the invention, unlike a single crystal structure, is able to integrate the nature and extent of the flexibility of the active site residues in the NNI binding site of RT. This uniquely permits prediction of potential modification sites on PETT, DABO, and HEPT derivatives after positioning the compounds in the NNI active site of RT. The method for designing new NNI compounds was particularly useful given the fact that no known crystal structures exist for RT-PETT and RT-DABO complexes, a fact which in this case would prevent the successful application of traditional structure-based drug design methods. Importantly, the model was validated by experimentally demonstrating the superior potency of newly designed agents, predicted to have strong RT inhibitory activity, based upon the low K_(i) values estimated.

Example 2 Predicted Efficacy of HEPT Derivatives

Compounds listed in Table 1 have been modeled into the NNI binding site of RT (RT/MKC 422 complex) using the docking procedure. The modeled positions were compared with the composite binding pocket of the invention, having the coordinates set forth in Table 9. Modeling was followed by analysis with the LUDI score function.

All of the positions of the compounds with top scores fall into the butterfly-shaped binding site, with the benzyl ring residing in wing 1 and the thymine ring in the wing 2 (FIG. 2). For all compounds tested, the benzyl ring is near Trp229 and the N-1 group is near Pro236, a typical position observed in crystal structures (FIG. 1B). The trend of calculated values listed in Table 1 shows that the K_(i) value decreases as a result of three factors: para substituents (R2) removed from the benzyl ring, larger alkyl groups added to the thymine ring (R₁), and sulfur atoms substituted for oxygen (at X and/or Y). The modeling calculations, along with the application of the composite NNI binding pocket, provided a guideline for the synthesis of lead compounds designed to have potent anti-HIV activity. The choice of compounds was also based on synthetic feasibility.

TABLE 1 Results of modeling calculations for HEPT derivatives

Accessible Molecular Buried LUDI LUDI Surface surface Surface Score Score^(d) Ki^(d) X Y R₁ R₂ R₃ NR^(a) (Å²) (Å²) (%) (Lipo) (Sum) (TM) O O Et F Et 6 549 296 n.d. n.d. n.d. n.d O O Et Br Et 6 576 311 n.d. n.d. n.d. n.d S O Me OMe Et 6 558 303 n.d. n.d. n.d. n.d O O Me H Et 5 505 269 85 599 463 23 O O Et H Et 6 528 284 87 661 501 9.8 O O i-Pr H Et 6 541 294 88 688 528 5.2 S O Me H Et 5 512 275 87 703 567 2.1 S O Et H Et 6 536 290 90 732 572 1.9 S O i-Pr H Et 6 550 300 89 741 580 1.5 S S Me H Et 5 521 283 86 706 570 2.0 S S Et H Et 6 545 297 90 756 595 1.1 S S i-Pr H Et 6 557 308 90 777 617 0.68 S S Me H Me 4 491 266 84 661 549 3.2 S S Et H Me 5 514 280 88 703 567 2.1 S S i-Pr H Me 5 527 290 90 738 602 0.95 Me = methyl, Et = ethyl, i-Pr = isoproyl n.d. (not determined) means high K_(i) values resulting from energetically unfavorable rotation of Trp 229 which sterically hinders binding in cases of the para substitution, as revealed by modeling. ^(a)NR = number of rotatable bonds in the compounds. Used in the LUDI calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom. ^(b)Molecular surface area calculated using the program GRASP, and defined as the boundary of the volume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with the hard sphere atoms which make up the molecule. The values are slightly smaller than the ones approximated by LUDI program. The accessible surface can be defined as the locus of the centers of all possible such probes in contact with the hard sphere atoms. Alternatively it can be defined as the hard sphere surface if each atomic radius is increased by the probe radius (1.4 Å radius). ^(c)Buried surface represents the percentage of molecular surface in contact with the protein calculated by LUDI based on the docked positions. Based on published crystal structures of RT complexes, the calculation shows that these values could be as low as 77% (in RT/HEPT complex) and can be as high as 90% (in RT/APA complex) but most of them including RT/MKC average around 84%. Therefore, the calculated values may be in the worst case slightly overestimated. ^(d)Ideal hydrogen bond distances and angles between the compounds and the protein are assumed in all cases for K_(i) and Score (sum) calculation. In published crystal structures of RT complexes, hydrogen bond geometry's are indeed close to ideal; the amide carbonyl of residue K101 on a loop demostrates a substantial flexibility which can accommodate the best geometry for hydrogen bonding.

Synthesis of HEPT Derivatives

The compounds listed in Table 1 above can be synthesized by reaction of substituted aryl acetonitriles and appropriately functionalized 2-bromo ethyl esters, for example in the presence of zinc in refluxing tetrahydrofuran. Products of the reaction are purified by gel chromatography. Generated 3-oxo esters are next converted into 5-alkyl-6-(arylmethyl)-2-thiouracils with chloroacetic acid, e.g., overnight to yield 5-alkyl-6-(arylmethyl)uracils. The final step in the synthesis is reaction of the uracil with hexamethyldisilazane (HMDS) in the presence of ammonium sulfate. Subsequent treatment with acetals and trimethyl silyl triflate in acetonitrile leads to the formation of N-′substituted uracil and thiouracil derivatives.

These and other known methods can be used to synthesize the compounds of the invention.

Example 3 DABO Derivatives

Chemical Synthesis

All chemicals were used as received from Aldrich Chemical Company (Milwaukee, Wis.). All reactions were carried out under nitrogen. Column chromatography was performed using EM Science silica gel 60 and one of the following solvents: ethyl acetate, methanol, chloroform, hexane, or methylene chloride. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian (Palo Alto, Calif.) 300 MHz instrument (Mercury 2000 model) and chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard at 0 ppm. ¹³C NMR spectra were recorded at 75 MHz in CDCl₃ on the same instrument using a proton decoupling technique. The chemical shifts reported for ¹³C NMR are referenced to the chloroform triplet at 77 ppm. Melting points were measured using a Mel-Temp 3.0 (Laboratory Devices Inc., Holliston, Mass.) melting apparatus and are uncorrected. UV spectra were recorded from a Beckmann (Fullerton, Calif.) model DU 7400 UV/Vis spectrometer using a cell path length of 1 cm and methanol solvent. Fourier Transform Infrared spectra were recorded using an FT-Nicolet (Madison, Wis.) model Protege 460 instrument. Mass spectrum analysis was performed using a Hewlett-Packard (Palo Alto, Calif.) Matrix Assisted Laser Description time-of-flight (MALDI-TOF) spectrometer (model G2025A) in the molecular ion detection mode (matrix used was cyanohydroxycinnamic acid). Some samples were analyzed using a Finnigan (Madison, Wis.) MAT 95 instrument. Elemental analysis was performed by Atlantic Microlabs (Norcross, Ga.).

General Procedure for the Synthesis of DABO Compounds 3a-d

The 5-alkyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-one derivatives 3a-d were prepared as shown in Scheme 1.

Ethyl-2-alkyl-4-(phenyl)-3-oxobutyrates 1a-d were obtained from commercially available phenyl acetonitrile. The β-ketoesters were condensed with thiourea in the presence of sodium ethoxide to furnish the corresponding thiouracils 2a-d. Compounds (1a-d and 2a-d) were produced by a methods previously described (Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med Chem., 1997, 40, 1447-1454; Danel, K. et al., J. Med. Chem., 1998, 41, 191-198).

Subsequent reaction of thiouracil with methylchloromethyl sulfide in N,N-dimethylformamide (DMF) in the presence of potassium carbonate afforded compounds 3a-d in moderate yields A mixture of thiouracil compound 2 (1 mmol), methylchloromethyl sulfide (1 mmol), and potassium carbonate (1 mmol) in anhydrous DMF (5 ml) was stirred overnight at room temperature. After treatment with water (50 ml), the solution was extracted with ethyl acetate (3×50 ml). The combined extracts were washed with saturated NaCl (2×50 ml), dried (MgSO₄), filtered and concentrated in vacuo to give the crude products 3a-d which were purified by column chromatography (hexane: ethyl acetate eluent).

X-ray Crystallography

Yellow rectangular plates of compound 3b were grown from tetrahydrofuran by slow evaporation at room temperature. X-ray diffraction data for a 0.5×0.2×0.08 mm plate crystal of compound 3b was collected at room temperature using a SMART CCD X-ray detector (Bruker Analytical X-ray Systems, Madison, Wis.). Structure solution and refinement was performed using the SHELXTL suite of programs (Bruker Analytical X-ray Systems, Madison, Wis.). All nonhydrogen atoms were refined using anisotropic displacement parameters. Hydrogen atoms were placed at ideal positions and refined as riding atoms with relative isotropic displacement parameters.

The refined small molecule X-ray crystal structure of compound 3b is shown as an Oak Ridge Thermal Ellipsoid Program (ORTEP) drawing in FIG. 8. Table 2 lists the crystal data and structure refinement statistics for compound 3b. Data was collected at room temperature (λ=0.71073 Å), refined using full-matrix least-squares refinement on F², and corrected for absorption using semi-empirical psi-scans.

TABLE 2 Unit Cell a = 4.7893(4) Å b = 10.8709(10) Å c = 30.040(3) Å α = 90° β = 92.474(2)° γ = 90° Space Group P2₁/n Unit Cell Volume 1562.5(2) Å³ Z 4 θ range for data collection 1.36 to 28.27° Limiting indices −6 ≦ h ≦ 6 −8 ≦ k ≦ 14 −39 ≦ l ≦ 37 Reflections collected 8744 Independent reflections 3507 (R_(int) = 0.0486) Data/restraints/parameters 3507/0/183 Goodness-of-fit on F² 1.095 Final R indices [I > 2σ(I)] R1 = 0.0666, wR2 = 0.1384 R indices (all data) R1 = 0.1114, wR2 = 0.1569 Absorption coefficient 0.338 mm⁻¹ Max. and min. transmission 0.8356 and 0.6542 Extinction coefficient 0.0004(11) Largest difference peaks 0.279 and −0.211 eÅ⁻³ R_(int) = Σ|F_(o) ² − <F_(o) ²>|/Σ|F_(o) ²|, R1 = Σ| |F_(o)| − |F_(c)| |/Σ|F_(o)| wR2 = {Σ[w(F_(o) ² − F_(c) ²)²]/Σ[w(F_(o) ²)²]}^(1/2) GooF = S = {Σ[w(F_(o) ² − F_(c) ²)²]/(n − p)}^(1/2), where n = reflections, p = parameters

Physical Data of Synthesized Compounds

5-methyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin4-1H-one (3a)

Yield 62%; mp 148-149° C.; ¹H NMR(CDCl₃): δ2.10 (s, 3H), 2.14 (s, 3H), 3.91 (s, 2H), 4.29 (s, 2H), 7.29-7.26 (m, 5H), 12.20 (s, 1H); ¹³C NMR(CDCl₃): δ10.7 (CH₃), 15.5 (SCH₃), 36.6 (CH₂Ph), 41.0 (SCH₂), 116.7 (C-5), 137.6-126.4 (Ph), 155.2 (C-6), 162.0 (C-4), 165.1 (C-2); Cl—MS: 293.1 (M+1).

5-ethyl-2-[(methylthiomethyl)thiol-6-benzyl-pyrimidin-4-1H-one (3b)

Yield 65%; mp 124-126° C.; ¹H NMR(CDCl₃): δ1.08 (t, 3H), 2.12 (s, 3H), 2.58 (q, 2H), 3.91 (s, 2H), 4.26 (s, 2H), 7.28-7.26 (m, 5H), 12.30 (s, 1H); ¹³C NMR(CDCl₃): δ13.1 (CH₃), 15.4 (SCH₃), 18.7 (CH₂), 36.4 (CH₂Ph), 40.3 (SCH₂), 122.4 (C-5), 138.0-126.3 (Ph),155.4 (C-6), 161.5 (C-4), 165.2 (C-2); CI—MS: 307.1 (M+1).

5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-1H-one (3c)

Yield 57%; mp 116-117° C.; ¹H NMR(CDCl₃): δ1.22 (d, 6H), 2.07 (s, 3H), 3.03 (q, 1H), 3.88 (s, 2H), 4.21 (s, 2H), 7.24-7.13 (m, 5H), 12.43 (s, 1H); ³C NMR(CDCl₃): δ15.4 (SCH₃), 19.6 (CH₃), 28.0 (CH), 36.3 (CH₂Ph), 40.9 (SCH₂), 125.3 (C-5), 138.3-126.3 (Ph), 155.5 (C-6), 161.1 (C-4), 164.5 (C-2); CI—MS 321.1 (M+1).

5-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl)-pyrimidin-4-1H-one (3d)

Yield 67%; mp 116-120° C.; ¹H NMR(CDCl₃): δ1.28 (d, 6H), 2.15 (s, 3H), 2.27 (s, 6H), 3.10 (q, 1H), 3.88 (s, 2H), 4.31 (s, 2H), 6.84 (s, 3H), 12.42 (s, 1H); ¹³C NMR(CDCl₃): δ15.3 (SCH₃), 19.6 (CH₃), 21.2 (CH₃), 28.0 (CH), 36.3 (CH₂Ph), 40.8 (SCH₂), 125.2 (C-5), 138.0-126.5 (Ph), 155.4 (C-6), 161.3 (C-4), 164.7 (C-2); CI—MS: 349.2 (M+1).

Modeling and Design of DABO Compounds

The calculated molecular coordinates of DABO compounds which were energy-minimized and docked into the NNI binding site adopted a conformation remarkably similar to that of the crystal structure of compound 3b. FIG. 7B shows the modeled coordinates superimposed on the crystal structure coordinates of 3b and illustrates their conformational similarity, suggesting that the final docked positions of the DABO compounds in the NNI pocket were energetically favorable and quite suitable for these studies. Multiple sterically allowed unoccupied spatial gaps in the binding site were identified from the docking studies which could be filled by strategically designed functional groups (FIG. 7B).

The docked DABO molecule (compound 3a) unexpectedly showed significant space surrounding the benzyl ring and the 5th position of the thymine ring, which led to design of compounds 3b, 3c and 3d. The inhibition constants of the docked molecules were calculated based on a LUDI score function and are listed in Table 3. The calculated K_(i) values suggested that compounds 3c and 3d would be particularly active inhibitors of RT.

Compound 3d, which differs from compound 3c by the addition of two methyl groups to the benzyl ring, provides more hydrophobic contact with the NNI binding pocket and was predicted to be more potent than compound 3c, based on the modeling studies. Calculations indicate that compounds 3a-3d have progressively larger molecular surface areas but still maintain approximately the same percentage of the molecular surface area in contact with the protein residues. Consequently, the calculated contact surface area between the protein and the compound increases in the following order: compound 3a, 3b, 3c, and 3d. This increased surface area in turn dictates a decrease in calculated K_(i) values, with 3a having the worst value and 3d the best.

The Tyr183 residue of the HIV RT is located in the catalytic region which has a conserved YMDD motif characteristic of reverse transcriptases. Therefore, the displacement of this tyrosine residue can interfere with catalysis and render the HIV-1 RT protein inactive. Bulky substituents at the 5th position of the thymine ring could indirectly accomplish such inactivation by displacing Tyr181 which is near Tyr183 (Ding, J. et al., Nat. Struct. Biol., 1995, 2, 407-415). The composite binding pocket shows sufficient room for at least a 3-carbon group at the 5th position. The addition of a methyl, ethyl or isopropyl group at the 5th position of the thymine ring is expected to lead to higher affinity for the relatively hydrophobic environment at this location of the binding pocket. The favorable hydrophobic contact increases as the hydrophobic group at the 5th position gets bulkier. As the DABO derivative binds to the site, the ethyl or isopropyl group can also cause the nearby Tyr181 residue to rotate away from the inhibitor.

Modeling studies showed that this change in conformation in turn affects the positions of neighboring Tyr183 and Tyr188 which may contribute to the inactivation of HIV-1 RT. The benzyl ring of compounds 3a-3d is located near a region surrounded by the hydrophobic ring planes of residues Trp229, Pro95, Y188 and Y181. The analysis of compounds 3a-3c in the composite binding pocket suggests that the benzyl ring would be located on the boundary of the pocket, near residue Y188. A para substituent of the ring is situated perpendicular to the ring plane of nearby Trp229, within van der Waals contact, and leaves a lot of space unfilled between the compound and Pro95. With a slight conformational rotation of the benzyl ring, compound 3d, with the addition of two methyl groups, was found to better fill the composite binding pocket (FIG. 7B). Such observations indicate that further modifications to the benzyl ring could lead to even more potent inhibitors.

TABLE 3 Dabo Compounds

Ludi^(a) Compound M.S.^(b) B.S.^(c) Lipo K_(j) Number R₁ R₂ (Å²) (%) Score (μM) 3a H Me 275 88 709 3.3 3b H Et 283 88 730 2.0 3c H i-Pr 301 89 785 0.56 3d Me i-Pr 329 89 875 0.05 ^(a)Ludi K_(j) values were calculated based on the empirical score function in Ludi program (Bohm, H. J., J. Comput. Aided. Mol. Des., 1994, 8, 243-256; 1996). Ideal hydrogen bond distances and angles between compounds and protein are assumed in all cases for Ludi K_(i) and Ludi Score calculation. In published crystal structures of RT complexes, hydrogen bond geometries are indeed close # to ideal; the amide carbonyl of residue K101 on a loop demonstrates substantial flexibility which can accommodate the best geometry for hydrogen bonding. The number of rotatable bonds (=2) is used in the Ludi calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom. ^(b)MS, molecular surface area calculated using Connolly's MS program (Connolly, M. L., Science, 1983, 221, 709-713). Defined as boundary of volume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with hard sphere atoms which make up the molecule. Values are slightly smaller than those approximated by Ludi program. ^(c)BS, buried surface: percentage of molecular surface in contact with protein calculated by Ludi relative to docked positions. Based on published crystal structures of RT complexes, the calculation shows that these values could be as low as 77% (in RT-HEPT complex) and can be as high as 90% (in RT-APA complex) but most of them including RT-MKC average around 84%.

Predictable Activities

The trend of the calculated K_(i) values based on the modeling and on the use of the composite binding pocket, with surprising accuracy, predicted the trend of the experimentally determined IC₅₀ values from HIV replication assays. Compounds 3a-3d were tested for RT inhibitory activity in cell-free assays using purified recombinant HIV RT (listed as IC₅₀[rRT] in Table 4), as well as by in vitro assays of anti-HIV activity in HTLVIIIB-infected peripheral blood mononuclear cells (IC₅₀[p24] in Table 4) (Zarling, J. M. et al., Nature, 1990, 347, 92-95; Erice, A. et al., Antimicrob. Ag. Chemother., 1993, 37, 835; Uckun, F. M. et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383).

Larger compounds which better fill the composite binding pocket and have lower calculated K_(i) values showed better IC₅₀[rRT] values. This is reflected by the enhancement of the inhibitory activity with the addition of progressively larger groups such as methyl (3a), ethyl (3b), and isopropyl (3c) at the C-5 position of the thymine ring (see Table 4). The same trend was also observed for IC₅₀[p24] values.

The lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)pyrimidin-4-(1H)-one (compound 3c), elicited potent anti-HIV activity with an IC₅₀ value less than 1 nM for inhibition of HIV replication (measured by p24 production in HIV-infected human peripheral blood mononuclear cells) and showed no detectable cytotoxicity (inhibition of cellular proliferation was >100 μM as measured by MTA) (Table 4). In contrast to all previously published data for DABO and S-DABO derivatives which were less active than AZT and MKC-442 (Danel, K. et al., Acta Chemica Scandinavica, 1997, 51, 426-430; Mai, A. et al., J. Med. Chem., 1997, 40, 1447-1454; Danel, K. et al., J. Med. Chem., 1998, 41, 191-198) and showed selectivity indices of <1,000, the novel compound 3c was more than 4-fold more active than AZT and MKC-442, and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations with an unprecedented selectivity index (=IC₅₀[MTA]/IC₅₀[p24]) of >100,000.

The X-ray crystal structure of 3b was determined to compare its conformation to that of the compound after docking into the NNI binding site. The refined small molecule X-ray crystal structure of compound 3b is represented as an ORTEP drawing in FIG. 8. The calculated molecular coordinates of DABO compounds which were energy-minimized and docked into the NNI binding site adopted a conformation remarkably similar to that of the crystal structure of compound 3b. FIG. 7B shows the modeled coordinates superimposed on the crystal structure coordinates of 3b and illustrates their conformational similarity, suggesting that the final docked positions of the DABO compounds in the NNI pocket were energetically favorable.

Table 4: Inhibitory Activity of DABO Compounds

TABLE 4 Inhibitory Activity of DABO Compounds

IC₅₀ IC₅₀ CC₅₀ Compound rRT] [p24] [MTA] Number R₁ R₂ (μM) (μM) (μM) S.I.^(d) 3a H Me 18.8 4.5 >100 >22 3b H Et 9.7 0.8 >100 >125 3c H i-Pr 6.1 <0.001 >100 >100,000 3d Me i-Pr 4.8 n.d. n.d. n.d. AZT >100 0.04 50 1250 MKC-442 0.004 >100 >25,000 ^(d)Selectivity Index is equal to the ratio of fifty percent cytotoxic concentration to IC₅₀. n.d. = not determined

Example 4 Synthesis of PETT Derivatives

Chemical Synthesis

All chemicals were used as received from Aldrich Chemical Company (Milwaukee, Wis.). All reactions were carried out under nitrogen. Column chromatography was performed using EM Science silica gel 60 and one of the following solvents: ethyl acetate, methanol, chloroform, hexane, or methylene chloride. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian (Palo Alto, Calif.) 300 MHz instrument (Mercury 2000 model) and chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard at 0 ppm. ¹³C NMR spectra were recorded at 75 MHz in CDCl₃ on the same instrument using a proton decoupling technique. The chemical shifts reported for ¹³C NMR are referenced to the chloroform triplet at 77 ppm. Melting points were measured using a Mel-Temp 3.0 (Laboratory Devices Inc., Holliston, Mass.) melting apparatus and are uncorrected. UV spectra were recorded from a Beckmann (Fullerton, Calif.) model DU 7400 UV/Vis spectrometer using a cell path length of 1 cm and methanol solvent. Fourier Transform Infrared spectra were recorded using an FT-Nicolet (Madison, Wis.) model Protege 460 instrument. Mass spectrum analysis was performed using a Hewlett-Packard (Palo Alto, Calif.) Matrix Assisted Laser Desorption time-of-flight (MALDI-TOF) spectrometer (model G2025A) in the molecular ion detection mode (matrix used was cyanohydroxycinnamic acid). Some samples were analyzed using a Finnigan (Madison, Wis.) MAT 95 instrument. Elemental analysis was performed by Atlantic Microlabs (Norcross, Ga.).

General Procedure for Synthesis of PETT Derivatives

Compounds I-1, I-3, and I-4 were synthesized as described in Scheme 3. Trovirdine (I-2) was synthesized according to the literature procedure (Bell, F. W., et al., J. Med. Chem., 1995, 38, 4929-4936).

Physical Data of Synthesized Compounds

N-[2-(2-pyridylethyl)]-N′-[2-(pyridyl)]-thiourea (I-1)

white solid (1 g, 49%); mp 98-100° C.; UV(MeOH) λmax: 293, 265, 247 and 209 nm; IR(KBr Disc) ν3415, 3222, 3050, 2360, 1600, 1533, 1479, 1436, 1315, 1240, 1151 and 775 cm⁻¹; ¹H NMR (CDCl₃) δ11.90 (s, 1H), 8.8 (s, 1H), 8.60-8.58 (d, 1H), 8.03-8.01 (d, 1H), 7.65-7.56 (m, 2H), 7.27-7.14 (m, 2H), 6.93-6.89 (d, 1H), 6.80-6.77 (d, 1H) 4.23-4.15 (q, 2H), and 3.41-3.20 (t, 2H); ¹³C NMR(CDCl₃) δ179.2, 158.9, 153.0, 149.2, 145.5, 138.5, 136.4, 123.5, 121.4, 117.7, 111.8, 44.9, and 36.9; MALDI-TOF mass found, 257.1(M−1), calculated, 258.3; Anal. (C₁₃H₁₄N₄S) C, H, N, S.

N-[2-(1-piperidinoethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (I-3)

white solid (2 g, 74%); mp 150-152° C.; UV (MeOH) λmax: 306, 275 and 205 nm; IR(KBr Disc) ν3155, 3077, 2935, 2850, 2360, 1591, 1525, 1465, 1319, 1226, 1095, 827 and 756 cm⁻¹; ¹H NMR (CDCl₃) δ8 11.53 (br s, 1H), 9.72 (br s, 1H), 8.22 (d, 1H), 7.72-7.68 (dd,1H), 6.95-6.92 (d, 1H), 3.84-3.78 (q, 2H), 2.61-2.57 (t, 2H), 2.45 (br s, 4H), 1.64-1.48 (m, 6H); ¹³C NMR(CDCl₃) δ178.1, 151.8, 146.3, 140.8, 113.5, 112.6, 56.1, 54.0, 43.0, 26.3, and 24.3, MALDI-TOF mass found, 343.5, calculated, 343.3; Anal. (C₁₃H₁₉BrN₄S) C, H, N, S, Br.

N-[2-(2,5-dimethoxyphenylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (I-4)

white solid (2 g, 67%); mp 133-138° C.; UV (MeOH) λmax: 202, 205, 231, 276 and 300 nm; IR(KBr Disc) ν3209, 3152, 3078, 3028, 2951, 2831, 1595, 1533, 1468, 1306, 1227, 1095, 1059, 1022, 862, 825, 796, 707 cm⁻¹; ¹H NMR(CDCl₃) δ11.24 (br s, 1H), 9.30 (br s, 1H), 8.10-8.09 (d, 1H), 7.65 (dd, 1H), 6.82-6.76 (m, 4H), 4.03-3.97 (q, 2H), 3.77 (s, 3H), 3.00-2.96 (t, 2H); ¹³C NMR(CDCl₃) δ178.7, 153.1, 151.8, 151.7, 146.5, 140.9, 128.1, 117.7, 113.3, 112.6, 111.2, 110.9, 55.7, 55.5, 45.6, and 29.9; MALDI-TOF mass found, 394.0 (M−1), 396.0 (M+1), calculated, 395.0; Anal. (C₁₆H₁₈BrN₃O₂S) C, H, N, S, Br.

Chemical Synthesis II

Compounds II-1-9 were synthesized according to Scheme 4. In brief, 2-amino-5-bromopyridine was condensed with 1,1-thiocarbonyl diimidazole to firnish the precursor thiocarbonyl derivative (A). Further reaction with appropriately substituted phenylethyl amine gave the target PETT derivatives in good yields.

General Procedure for Synthesis

Thiocarbonyldiimidazole (8.90 g, 50 mmol) and 2-amino-5-bromo pyridine (8.92 g, 50 mmol) were added to 50 mL of dry acetonitrile at room temperature. The reaction mixture was stirred for 12 h and the precipitate filtered, washed with cold acetonitrile (2×25 mL), and dried under vacuum to afford (11.40 g, 80% ) of compound A. To a suspension of compound A (0.55 eqv) in dimethyl formamide (15 mL) an appropriate amine (0.50 eqv) was added. The reaction mixture was heated to 100° C. and stirred for 15 hours. The reaction mixture was poured into ice-cold water and the suspension was stirred for 30 minutes. The product was filtered, washed with water, dried, and further purified by column chromatography to flimish the target compounds 1-9 in good yields.

Peagents and conditions: 1) 1,1-thiocarbonyldiimidazole, acetonitrile, RT, 12h.

Physical Data of Synthesized Compounds

N-[2-(2-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-1)

yield: 65%; mp 143-145° C.; UV (MeOH) λmax: 202, 205, 275 and 306 nm; IR(KBr) ν3211, 3153, 3036, 2956, 2835, 1593, 1533, 1462, 1242, 1186, 1036, 1007, 862, 812, 756, 708 cm⁻¹; ¹H NMR (CDCl₃) δ11.22 (br s, 1H), 9.37 (br s, 1H), 8.02-8.01 (d, 1H), 7.69-7.65 (dd, 1H), 7.28-7.18 (m, 2H), 6.94-6.80 (m, 3H), 4.04-3.98 (q, 2H), 3.81 (s, 3H), 3.04-2.99 (t,2H), ¹³C NMR(CDCl₃) δ178.7, 157.6, 151.7, 146.3, 141.0, 130.7, 127.9, 126.8, 120.3, 113.5, 112.5, 110.3, 55.2, 45.6, 29.8; Maldi Tof found: 366.0 (M+1), calculated: 365.0; Anal. (C₁₅H₁₆BrN₃OS) C, H, N, S.

N-[2-(2-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-2)

yield: 71%; mp 156-157° C.; UV (MeOH) λmax: 209, 256, 274 and 305 nm; IR(KBr) ν3446, 3234, 3163, 3055, 2935, 1672, 1595, 1560, 1531, 1466, 1390, 1362, 1311, 1265, 1227, 1169, 1136, 1089, 1003, 864, 825, 756 cm⁻¹; ¹H NMR (CDCl₃) δ11.36 (br s, 1H), 9.47 (br s, 1H), 8.05-8.04 (d, 1H), 7.72-7.68(dd, 1H), 7.30-7.03 (m, 4H), 6.87-6.84 (d, 1H), 4.06-3.99 (q, 2H), 3.10-3.05 (t, 2H); ¹³C NMR(CDCl₃) δ179.1, 163.1, 151.7, 146.2, 141.1, 131.2, 131.1, 128.5, 128.4, 124.1, 115.5, 115.2, 113.6, 112.2, 45.8 and 28.2; ¹⁹F NMR(CDCl₃) δ−42.58 & −42.55 (d); Maldi Tof found: 355.0 (M+1), calculated: 354.0; Anal. (C₁₄H₁₃BrFN₃S) C, H, N, S.

N-[2-(2-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-3)

yield: 72%; mp 137-139° C.; UV (MeOH) λmax: 208, 213, 256, 275 and 305 nm; IR(KBr) ν3433, 3221, 3157, 3089, 3037, 2922, 2866, 1668, 1597, 1535, 1466, 1338, 1263, 1209, 1188, 1130, 1095, 1053, 1001, 864, 823, 750 cm⁻¹; ¹H NMR (CDCl₃) δ11.41 (br s, 1H), 9.54 (br s, 1H), 8.17-8.16 (d, 1H), 7.83-7.79 (dd, 1H), 7.50-7.30 (m, 4H), 6.97-6.94 (d, 1H), 4.19-4.13 (q, 2H), 3.30-3.26(t, 2H); ¹³C NMR(CDCl₃) δ179.2, 151.7, 146.3, 141.2, 136.3, 134.2, 131.1, 129.6, 128.1, 126.8, 113.6, 112.7, 45.2, and 32.5; Maldi Tof found: 371.8 (M+l), calculated: 371.0; Anal. (C₁₄H₁₃BrClN₃S) C, H, N, S, Br.

N-[2-(3-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-4)

yield: 68%; mp 155-156° C.; UV (MeOH) λmax: 208, 274 and 306 nm; IR(KBr) ν3454, 3236, 3147, 3030, 2951, 2869, 2827, 1591, 1545, 1525, 1466, 1304, 1265, 1229, 1188, 1151, 1095, 1051, 1024, 980, 860, 825, 789, 698 cm⁻¹; ¹H NMR (CDCl₃) δ11.30 (br s, 1H), 9.25 (br s, 1H), 8.05-8.04 (d, 1H), 7.71-7.67 (dd, 1H), 7.29-7.24 (t, 1H), 6.89-6.78 (m, 4H), 4.05-3.99 (q, 2H), 3.81 (s, 3H), 3.00-2.96 (t, 2H); ¹³C NMR(CDCl₃) δ178.9, 159.7, 151.6, 146.4, 141.1, 140.3, 129.6, 121.2, 115.0, 113.4, 112.7, 111.6, 55.1, 47.1 and 34.8; Maldi Tof found: 367.0 (M+2), calculated: 365.0; Anal. (C₁₅H₁₆BrN₃OS) C, H, N, S.

N-[2-(3-fluorophenethyl)]-N′-12-(5-bromopyridyl)]-thiourea (II-5)

yield: 73%; mp 171-172° C.; UV (MeOH) λmax: 202, 208, 258, 275 and 306 nm; IR(KBr) ν 3213, 3155, 3084, 3028, 2866, 1595, 1533, 1477, 1336, 1308, 1229, 1211, 1173, 1136, 1092, 1026, 935, 870, 827, 791, 740 cm⁻¹; ¹H NMR (CDCl₃) 611.33 (br s, 1H), 9.46 (br s, 1H), 8.05-8.04 (d, 1H), 7.73-7.69 (dd, 1H), 7.31-7.26 (m, 1H), 7.08-6.97 (m, 3H), 6.87-6.83 (d, 1H), 4.06-3.99 (q, 2H), 3.05-3.00 (t, 2H); ¹³C NMR (CDCl₃) δ179.1, 163.1, 151.7, 146.2, 141.2, 130.1, 129.9, 124.5, 115.9, 115.6, 113.7, 113.5, 113.4, 112.8, 46.7 and 34.6; ¹⁹F NMR(CDCl₃) δ−37.30 & −37.33 (d); Maldi Tof found: 354.0 (M⁺), calculated: 354.0; Anal. (C₁₄H₁₃BrFN₃S) C, H, N, S.

N-[2-(3-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-6)

yield: 72%; mp 163-165° C.; UV (MeOH) λmax: 202, 213, 258, 276 and 305 nm; IR(KBr) ν 3242, 3161, 3043, 2929, 1593, 1579, 1547, 1527, 1466, 1313, 1227, 1167, 1095, 997, 889, 827, 812, 785, 700 cm⁻¹; ¹H NMR (CDCl₃) δ11.33 (br s, 1H), 9.37 (br s, 1H), 8.09-8.08 (d, 1H), 7.73-7.69 (dd, 1H), 7.28-7.15 (m, 4H), 6.85-6.82 (d, 1H), 4.04-3.98 (q, 2H), 3.02-2.97 (t, 2H), ¹³C NMR (CDCl₃) δ179.1, 151.6, 146.3, 141.2, 140.7, 134.2, 129.8, 129.0, 127.0, 126.8, 113.4, 112.8, 46.7 and 34.5; Maldi Tof found: 371.8 (M+1), calculated: 371.0; Anal. (C₁₄H₁₃BrClN₃S) C, H, N, S.

N-[2-(4-methoxyphenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-7)

yield: 85%; mp 178-179° C.; UV (MeOH) λmax: 205, 226, 275 and 305 nm; IR(KBr) ν3221, 3159, 3042, 2931, 2827, 1587, 1510, 1464, 1311, 1225, 1165, 1088, 1034, 820, 773, 708 cm⁻¹; ¹H NMR (CDCl₃) δ11.30 (br s, 1H), 9.87 (br s, 1H), 8.00-7.99 (d, 1H), 7.67-7.63 (dd, 1H), 7.21-7.18 (d, 2H), 6.95-6.85 (m, 3H), 4.00-3.93 (q, 2H), 3.81 (s, 3H), 2.96-2.92 (t, 2H), ¹³C NMR (CDCl₃) δ179.1, 158.0, 151.9, 145.8, 140.7, 130.6, 129.6, 113.8, 113.7, 112.1, 55.1, 46.9 and 33.8; Maldi Toffound: 366.0 (M+1), calculated: 365.0; Anal. (C₁₅H₁₆BrN₃OS) C, H, N, S.

N-[2-(4-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-8)

yield: 69%; mp 177-178° C.; UV (MeOH) λmax: 208, 211, 274 and 306 nm; IR(KBr) ν3456, 3213, 3155, 3086, 3028, 2868, 1595, 1560, 1533, 1477, 1336, 1308, 1238, 1211, 1173, 1136, 1092, 1026, 933, 869, 827, 791, 741, 694 cm⁻¹; ¹H NMR (CDCl₃) δ11.29 (br s, 1H), 9.27 (br s, 1H), 8.04-8.03 (d, 1H), 7.73-7.69 (dd, 1H), 7.27-7.22 (m, 2H), 7.04-6.99 (m, 2H), 6.83-6.79 (d, 1H), 4.03-3.96 (q, 2H), 3.02-2.97 (t, 2H); ¹³C NMR(CDCl₃) δ179.1, 163.2, 151.6, 146.3, 141.2, 134.3, 130.3, 130.2, 115.4, 115.2, 113.5, 112, 47.0, and 34.1; ¹⁹F NMR (CDCl₃) δ−40.55 (m); Maldi Tof found: 354.8 (M+1), calculated: 354.0; Anal. (C₁₄H₁₃BrFN₃S) C, H, N,S.

N-[2-(4-chlorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (II-9)

yield: 71%; mp 180-183° C.; UV (MeOH) λmax: 206, 209, 219, 256, 275 and 305 nm; IR(KBr) ν 3221, 3153, 3086, 3022, 2931, 1674, 1593, 1562, 1533, 1473, 1406, 1340, 1304, 1265, 1227, 1169, 1138, 1092, 1016, 820, 752, 714 cm⁻¹; ¹H NMR (CDCl₃) δ11.40 (br s, 1H), 9.34 (br s, 1H), 8.15-8.14 (d, 1H), 7.84-7.80 (dd, 1H), 7.46-7.30 (m, 4H), 6.92-6.89 (d, 1H), 4.10-4.07 (q, 2H), 3.13-3.08 (t, 2H); ¹³C NMR (CDCl₃) δ179.2, 151.6, 146.3, 141.3, 137.1, 130.2, 128.6, 113.5, 112.8,46.8 and 34.2; Maldi Tof found: 372.0 (M+1), calculated: 371.0; Anal. (C₁₄H₁₃BrClN₃S) C, H, N, S.

Chemical Synthesis III

Compounds III-1-3 were prepared as illustrated in Scheme 5. The synthesis involved condensing 2-amino-5-bromopyridine with 1,1-thiocarbonyl diimidazole to furnish the required thiocarbonyl derivative. Further reaction of this thiocarbonyl derivative with an appropriate amine gave 1-3 in good yields.

Scheme 5

Physical Data of Synthesized Compounds

N-[2-(1-piperidinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (III-1)

Yield: 74%; mp 150-152°; ¹H NMR (CDCl₃) δ11.53 (br s, 1H), 9.72 (br s, 1H), 8.22 (d, 1H), 7.72-7.68 (dd, 1H), 6.95-6.92 (d, 1H), 3.84-3.78 (q, 2H), 2.61-2.57 (t, 2H), (br s, 4H), 1.64-1.48 (m, 6H); ¹³C NMR(CDCl₃) δ178.1, 151.8, 146.3, 140.8, 113.5, 112.6, 56.1, 54.0, 43.0, 26.3, and 24.3.

N-[2-(1-piperizinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (III-2)

Yield: 75%; mp 178-180° C.; ¹H NMR (CDCl₃) δ11.50 (br s, 1H), 9.77 (br s, 1H), 8.19-8.18 (d, 1H), 7.75-7.71 (dd, 1H), 6.97-6.95 (d, 1H), 3.87-3.86 (m, 2H), 3.63-3.60 (t, 2H), 3.45-3.42 (m, 3H), 2.74-2.69 (t, 2H), 2.59-2.52 (m, 4H); ¹³C NMR(CDCl₃) δ178.7, 151.8, 146.1, 141.0, 113.7, 112.7, 55.2, 52.0, 51.9 and 45.8.

N-[2-(1-morpholinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (III-3)

Yield: 65%; 124-126° C.; ¹H NMR (CDCl₃) δ11.51 (br s, 1H), 9.23 (br s, 1H), 8.25-8.24 (d, 1H), 7.75-7.71 (dd, 1H), 6.85-6.82 (d, 1H), 3.87-3.74 (m, 6H), 2.68-2.54 (m, 6H); ¹³C NMR(CDCl₃) δ178.5, 151.7, 146.4, 141.0, 113.5, 112.7, 67.2, 55.4, 53.1, 42.5.

Compound R Compound R I-1 pyridyl II-1 piperidinyl I-3 piperidinyl III-2 piperozinyl I-4 2,5-dimethoxy phenyl III-3 morpholinyl II-1 o-methoxy phenyl II-6 m-chlorophenyl II-2 o-fluorophenyl II-7 p-methoxy phenyl II-3 o-chlorophenyl II-8 p-flurophenyl II-4 m-methoxy phenyl II-9 p-chlorophenyl II-5 m-fluorophenyl

Example 5 Structure-based Design and Docking of Novel PETT Derivatives into Composite NNI Binding Pocket I

A novel model of the NNI binding pocket of RT was constructed by carefully superimposing the coordinates of 9 individual RT-NNI crystal structures and then generating a van der Waals surface which encompassed all of the overlaid ligands. The integrated structural information of this “composite binding pocket” revealed an unexpectedly different and much larger NNI binding site than shown in or predictable from any of the individual structures and served as a probe to more accurately define the potentially usable space in the binding site (FIG. 2a). A number of protein residues in contact with the inhibitors are relatively flexible and vary from structure to structure. These residues include Tyr188, Tyr181, Tyr318, Try319, Phe227, Leu234, Trp229, Pro95, and Glu138 (from p51subunit of RT) (SEQ ID NO:1). As shown in FIG. 2b, the surface of the composite binding pocket is a short distance away from (<1.5 Å) or even extends past RT residues 234-236, Y188, F227, and the backbone of K101. This indicates that these residues are flexible and can be displaced by the right inhibitor. The composite binding pocket, unlike an individual crystal structure, is able to summarize the nature and extent of the flexibility of the active site residues. This allowed prediction of potential modification sites on the PETT derivatives I after positioning the compounds in the RT active site (see Methods).

A computer simulation of the binding of PETT compounds into the NNI binding site of RT was accomplished using a molecular docking procedure. Docking of PETT and trovirdine into the NNI binding site required the use of X-ray coordinates of an RT/NNI complex (in this case the RT/9-Cl-TIBO complex).

Upon binding to RT, the compound can fit into a butterfly-shaped NNI binding site (described by Ding, J., et al., Nat. Struct. Biol., 1995, 2, 407-415) (FIGS. 1B and 2). PETT and its derivatives such as compounds I-1-4 could be viewed as two chemical groups linked together by a thiourea group (Table 5). One half of the molecule is composed of a 2-aminothiazole group (PETT) or a pyridylthiourea group (compounds I-1-4) which forms an intramolecular hydrogen-bonded heterocyclic ring. The other half of the molecule is a phenyl or heterocyclic ring separated from the thiocarbonyl group by an ethyl linker.

Once the final docked position of the molecule in the NNI site was determined, the molecule was assigned a score, from which an estimation of the inhibition constant (K_(i) value) was determined (Table 5). When trovirdine was docked into the NNI binding site of RT it had a higher binding score than PETT and fit into the butterfly-shaped binding region with one part residing in Wing 1 and the other in Wing 2 (FIG. 1B). The ring closest to the thiocarbonyl group resided near the Lys(K)101 loop and the other pyridyl ring was near Trp(W)229.

After docking and K_(i) estimation was completed for the PETT inhibitors, evaluation of the docked compounds in the active site of RT involved placing each compound into the composite binding pocket using the same orientation matrix utilized in its construction. The potentially flexible regions in the binding site were then readily identified as were atom sites for future derivatization of the compounds. The area within Wing 2 and the residues near the thiourea group seemed to be the most forgiving regions in the binding site of RT. This observation was also supported by the analysis of gaps in atom-to-atom contact between the protein and the inhibitor.

TABLE 5 Interaction scores, calculated K_(i) values and measured IC₅₀ data for PETT derivatives I.

PETT I-1 to I-4 Ludi^(c) IC₅₀ M.S.^(a) B.S.^(b) Lipo Ludi^(c) K_(i) p24 R₁ R₂ (Å²) (%) Score Score (μM) (μM) S.I.^(d) PETT phenyl 2-thiazolyl 254 84 625 562 2.4 n.d. n.d I-1 2-pyridyl 2-pyridyl 260 84 640 577 1.7 0.230 >435 I-2 2-pyridyl 2-(5-bromo) 276 84 679 616 0.7 0.007 >10⁴ Trovirdine 2-pyridyl I-3 1-piperidinyl 2-(5-bromo) 278 84 684 621 0.6 <0.001 >10⁵ pyridyl I-4 2,5- 2-(5-bromo) 317 84 779 716 0.2 <0.001 >10⁵ dimethoxy- pyridyl phenyl AZT 0.008 6250 ^(a)MS, molecular surface area calculated using Connolly's MS program. (Connolly, M. L.; Science, 1983, 221, 709-713) Defined as boundary of vloume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with hard sphere atoms which make up the molecule. Values are slightly smaller than those approximated by Ludi program. ^(b)BS, buried surface: percentage of molecular surface in contact with protein calculated by Ludi based on docked positions. Based in published crystal structures of RT complexes, our calculation shows that these values could be as low as 77% (in RT-HEPT complex) and can be as high as 90% (in RT-APA complex) but most of them including RT-MKC average around 84%. ^(c)Ludi Ki values were calculated based on the empirical score function in Ludi program. (Bohm, H. J., J. Comput. Aided. Mol. Des., 1994, 8, 243-256; 1996,) Ideal hydrogen bond distances and angles between compounds and protein are assumed in all cases for Ludi K_(i)and Ludi Score calculation. In published crystal structures of RT complexes, hydrogen bond geometries are indeed close to ideal; the amide carbonyl of residue K101 on a loop demonstrates substantial flexibility which can # accommodate the best geometry for hydrogen bonding. The number of rotatable bonds(= 2) is used in the Ludi calculation to reflect the loss of binding energy due to freezing of internal degrees of freedom. ^(d)Selectivity Index is equal to the ratio of fifty percent cytotoxic concentration to IC₅₀. n.d., not determined.

Analysis of the molecular surface of the compounds in the NNI binding site of RT included visualization of spatial gaps between the compounds and nearby residues of the RT protein, as described above for Example 1. The spheres generated are shown in FIG. 3, and indicate regions of the compound which are probably not available for derivatization. FIG. 4 shows the binding pocket embellished with a grid of red rods which represent unoccupied space between the compound and active site residues, providing a complementary view to that shown by the spheres in FIG. 3. The grid illustrates the candidate sites for derivatization of the compound and, when used as a distance scale (the length of one rod represents 1 Å), also indicates the volume available for new functional groups. After the docked PETT compounds were subjected to the grid (gap) analysis, a number of gaps in the binding site were identified (FIGS. 3-4), some of which could be filled by strategically designed functional groups on new PETT derivatives. It was postulated that a more efficient use of such sterically allowed unoccupied spatial gaps in the binding site could be achieved by replacing the 2-pyridyl ring of trovirdine with a 1-piperidinyl (compound I-3) or 2,5-dimethoxyphenyl moiety (compound I-4) and yield potentially more active PETT compounds with larger molecular surface areas, higher Ludi scores, and lower K_(i) values (Table 5).

Compounds I-1, I-3 and I-4 were subjected to the same docking procedure and K_(i) calculation used to analyze the parent compounds PETT and trovirdine (compound I-2). The molecular surface area of the compounds calculated after docking increased in the following order: PETT, compound I-1, I-2 (trovirdine), I-3, and I-4. At docked positions, the atom surface area in contact with the protein residues constituted an average of 84% of the entire molecular surface (FIG. 3). We used this average value in the calculation of the inhibitory constant (K_(i)) based on the Ludi score function. Calculated Ki values for I-3 and I-4 predicted that these compounds would have potency superior to that of trovirdine. The calculated K_(i) values of our compound I-3 (0.6 μM), and compound I-4 (0.2 μM) were better than those of known compounds PETT (2.4 μM), compound I-1 (1.7 μM) and trovirdine (0.7 μM).

Example 6 In virto Assays of Anti-HIV Activity Using PETT Derivatives I

The HIV-1 strain HTLVIIIB (kindly provided by Dr. Neal T. Wetherall, VIROMED Laboratories, Inc.), was propagated in CCRF-CEM cells, and used in in vitro assays of the anti-HIV-1 activity of the synthesized novel derivatives. Cell-free supernatants of HTLVIIIB-infected CCRF-CEM cells were harvested, dispensed into 1 ml aliquots, and frozen at −70° C. Periodic titration of stock virus was performed by examining its cytopathic effects in MT-2 cells following the procedures described in (Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835).

Normal human peripheral blood mononuclear cells (PBMNC) from HIV-negative donors were cultured 72 hours in RPMI 1640 supplemented with 20%(v/v) heat-inactivated fetal bovine serum (FBS), 3% interleukin-2, 2 mM L-glutamine, 25 mM HEPES, 2 g/L NaHCO₃, 50 μg/ml gentamicin, and 4 μg/ml phytohemagglutinin prior to exposure to HIV-1. The incubated cells were then exposed to HIV-1 at a multiplicity of infection (MOI) of 0.1 during a one-hour adsorption period at 37° C. in a humidified 5% CO₂ atmosphere. Subsequently, infected cells were cultured in 96-well microtiter plates (100 μl/well; 2×10⁶ cells/ml) in the presence of test compounds, including AZT as a control. Aliquots of culture supernatants were removed from the wells on the 7th day after infection for p24 antigen assays. The methods used in the P24 assay were as previously described in Uckun, et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383; Zarling, et al., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835.

The applied p24 enzyme immunoassay (EIA) was the unmodified kinetic assay commercially available from Coulter Corporation/Immunotech, Inc. (Westbrooke, Me.). In the assay, a murine monoclonal antibody against HIV core protein is coated onto microwell strips. Antigen (HIV core protein) present in the test culture supernatant samples binds the antibody and the bound antibody-antigen complex is quantitated. Percent viral inhibition was calculated by comparing the p24 values from the test substance-treated infected cells with p24 values from untreated infected cells (i.e., virus controls).

In addition, the activity of the test compounds to inhibit recombinant HIV-1 reverse transcriptase (rRT) activity was determined using the Quan-T-RT assay system (Amersham, Arlington Heights, Ill.), which utilizes the scintillation proximity assay principle. The assay method is described in Bosworth, N., et al., Nature, 1989, 341, 167-168. Data for both bioassays is reported as IC₅₀ values.

In parallel with the bioactivity assays, the effects of the test compounds on cell viability was also examined, using the Microculture Tetrazolium Assay (MTA) described in Darling, et al., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag Chemother., 1993, 37, 835. In brief, non-infected PBMNC were treated with test compounds or controls for 7 days under identical experimental conditions and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), was added to quantitative cellular proliferation.

An energy-minimized model of compound I-4 in the RT binding site had the largest molecular surface area in contact with the protein and thus achieved the highest lipophilicity score. The docking studies indicated that the 2-methoxy group of compound I-4 is situated beneath the ethyl linker and fits favorably into a cavity of the binding pocket, providing contact with protein residues that cannot be achieved by trovirdine. Likewise, the 5-methoxy group of compound I-4 provides close contact with residues Pro95 and Trp229. The trend of the calculated K_(i) values accurately predicted the trend of the experimentally determined IC₅₀ values from HIV replication assays, as shown in Table 5, thereby providing conclusive evidence of the practical utility of the composite model.

The lead compound, I-4 with the lowest calculated K_(i) values of the series, was 8-times more potent than trovirdine against purified recombinant HIV-RT using the cell-free Quan-T-RT system (IC50[rRT] was 0.1 μM for I-4 versus 0.8 μM for trovirdine). Compound I-4 also elicited potent anti-HIV activity with IC₅₀ values of less than 0.001 μM in 3 of 3 independent experiments which was consistently lower than the IC₅₀ values for trovirdine (0.007 μM) and AZT (0.008 μM). None of the PETT derivatives were cytotoxic at concentrations as high as 100 μM. Therefore, the calculated selectivity index (IC₅₀[MTA]/IC₅₀[p24]) of compounds I-3 and I-4 were greater than 10⁵.

All active PETT compounds listed in Table 5 are able to form an intramolecular hydrogen bond between the nitrogen atom of pyridine or thiazole and an amide hydrogen of the thiourea group, as shown in Wing 1 of FIG. 1B. The intramolecular hydrogen bond was also observed in our small molecule crystal structure of compound I-3 (data not shown). The energy gained by the formation of such a hydrogen bond has been estimated to be about 5 kcal/mol (Bell, et al., J. Med. Chem., 1995, 38, 4929-4936). Our docking results showed that the internal hydrogen bond keeps the pyridyl thiourea (or thiazolylthiourea) in a more rigid conformation and allows the molecule to adopt the appropriate geometry to occupy Wing 1 of the binding site, and at the same time maintain a hydrogen bond with a backbone carbonyl of residue Lys101 (FIG. 1B).

Compounds I-3 and I-4 differ from trovirdine at the proposed Wing 2 binding region of the molecule. Compound I-3 has a heterocyclic ring which replaces the pyridyl ring and compound 4 has two methoxy groups added at meta and ortho positions of the phenyl ring. The molecular surface areas of compounds I-3 and I-4 are larger than that of trovirdine, as calculated from the coordinates of the predicted active conformation obtained from docking. This larger surface area results in a better lipophilic score and lower calculated K_(i) value (Table 5). Both pyridylethyl and piperidinylethyl groups occupy the same region of Wing 2 near Trp229 (FIG. 2 and 5). Our composite binding pocket shows a space large enough to accommodate a group larger than the pyridyl ring of trovirdine. Docking results and analyses of gaps indicate that the pyridyl ring of trovirdine has multiple sites which can be used for incorporation of larger groups. As shown in FIG. 5, there is sufficient space surrounding the pyridylethyl ring for the addition of a two- to four-atom substituent at any of the ring positions. Both sides of the pyridylethyl ring plane of trovirdine are relatively exposed in the pocket (FIG. 3A) and can accommodate additional substituents (FIG. 4A). This prediction was confirmed by the potency of compound I-4 (which contains ortho, meta-dimethoxy substituents), in inhibiting HIV replication.

The piperidinyl group of 1-3 is puckered and therefore occupies a larger overall volume than the planar pyridyl ring of trovirdine and is in close contact with residues Leu234 and Leu100, the latter of which can mutate to isoleucine, frequently found in a drug-resistant RT mutant strain. In contrast to previously reported extensive attempts at expanding within the pyridyl ring plane (Bell, et al., J. Med Chem., 1995, 38, 4929-4936; Cantrell, A. S., et al., J. Med. Chem., 1996, 39, 4261-4274; Ahgren, C., et al., Antimicrob. Agents Chemotherapy, 1995, 39, 1329-1335), the success of our efforts at modification perpendicular to the ring plane introduces new possibilities to develop more potent inhibitors which combine both modifications. The piperidinyl ring is conformationally more flexible than an aromatic ring has the advantage of fitting an uncompromising binding pocket more effectively, despite the expense paid for loss of entropy upon binding. The analysis shown in FIGS. 3, 4, and 5 provides new insights for modifications which are different from those of trovirdine derivatives. Various combinations of double substitutions at axial or equatorial positions of the piperidinyl ring generate derivatives with a broader range of curvatures than trovirdine derivatives and better fit Wing 2 which itself contains some curvature.

In summary, a composite binding pocket was constructed which integrated all available crystal structure information about the NNI binding site of RT. This novel computer-generated model was an unexpectedly effective tool that helped to much better comprehend the flexible nature of the binding pocket and to identify specific areas for structural improvements of the inhibitors. Nine lead NNI compounds from published crystal structures were analyzed. With all strategies combined, a number of previously unknown candidate sites for developing more potent derivatives of PETT were identified, such as substituting a bulkier piperidinyl group or an ortho/meta substituted phenyl group in place of an unsubstituted ring which resulted in enhanced inhibitory activity. The presented experimental results demonstrate that two novel PETT derivatives which resulted from our structure-based design efforts using the composite binding pocket are remarkably potent and noncytotoxic anti-HIV agents with unprecedented selectivity indices of >10⁵. The superior activity of these designed PETT compounds would not have been predictable from existing information about trovirdine alone, or from any single crystal structure of RT complexed with an NNI.

Example 7 Structure-based Design and Docking of PETT Derivatives into Composite NNI Binding Pocket II

The PETT derivatives II, synthesized as described above for Example 4, were analyzed for fit into the NNI binding pocket. Target compounds were also analyzed for anti-viral activity in p24 enzyme immunoassays and also for the ability to inhibit HIV reverse transcriptase activity, using rRT. Methods for these biological assays are described above for Example 6.

A computer simulation of the binding of the target PETT derivatives into the NNI binding site of RT was accomplished using a molecular docking procedure. Docking of the compounds into the NNI binding site required the use of X-ray coordinates of an RT/NNI complex (in this case the RT/9-Cl-TIBO complex).

Trovirdine derivatives could be viewed as two chemical groups linked together by a thiourea group (Table 6). One half of the molecule is composed of a pyridylthiourea group (compounds II-1-9) which forms an intramolecular hydrogen-bonded heterocyclic ring (shown in trovirdine structure). The other half of the molecule is a pyridyl ring separated from the thiocarbonyl group by an ethyl linker.

When trovirdine was docked into the NNI binding site of RT, it fit into the butterfly-shaped binding region (described by Ding, et al., Nat. Struct. Biol., 1995, 2, 407-415) with one part of the molecule residing in Wing 1 and the other in Wing 2. The ring closest to the thiocarbonyl group resided near the Lys(K)101 loop and the other pyridyl ring was near Trp(W)229.

Compounds II-1-9 were positioned according to this binding mode into the RT/9-Cl-TIBO active site by a docking procedure described above for Example 1. The results are shown in FIG. 6. One of the NH groups of the thiourea part of these compounds consistently formed a hydrogen bond with the backbone of K101.

Once the final, energetically favored docked position of the molecule in the NNI site was determined, a LUDI score was assigned, from which an estimation of the inhibition constant (K_(i) value) was determined (Table 6). The calculated K_(i) values, ranging from 0.4 μM to 0.8 μM suggested that compounds II-2 to II-7 would be active inhibitors of RT. The modeling data, shown below in Table 6, predicted that compounds II-2 to II-7 would be as potent as or more potent than trovirdine for inhibiting RT. The data for the bioassay of RT inhibition follows this prediction.

TABLE 6 Interaction scores, K_(i) values, and measured IC₅₀ data for a series of PETT derivatives.

II-1 to II-9 K_(i) IC₅₀ IC₅₀ Com- MS^(a) BS^(b) LIPO (calc) rRT* p24 pound X (Å²) (%) Score (μM)^(c) (μM) (μM) SI^(d) II-1 o-OMe 282 82% 678 1.2 1.0 0.01 >1 × 10⁴ II-2 o-F 281 82% 674 0.8 0.6 <0.001 >1 × 10⁵ II-3 o-Cl 285 83% 694 0.5 0.7 <0.001 >1 × 10⁵ II-4 m-OMe 296 84% 729 0.4 0.4 0.003 >3 × 10⁴ II-5 m-F 282 83% 687 0.6 0.7 <0.001 >1 × 10⁵ II-6 m-Cl 283 81% 672 0.8 3.1 N.D. N.D. II-7 p-OMe 302 83% 734 0.6 0.9 0.015 >6 × 10³ II-8 p-F 284 81% 674 7.8 6.4 N.D. N.D. II-9 p-Cl 293 81% 696 4.7 2.5 N.D. N.D. trovir- N.A. 276 84% 679 0.7 0.8 0.007 >1 × dine 10⁴ AZT N.A. N.A. N.A. N.A. N.A. >100 0.004 7 × 10³ •rRT, recombinant HIV reverse transcriptase assay ^(a)MS, molecular surface area calculated using Connolly's MS program.(Connolly, Science, 1983, 221, 709-713) Defined as boundary of volume within any probe sphere (meant to represent a water molecule) of given raduis sharing no volume with hard sphere atoms which make up the molecule. Values are slightly smaller than those approximated by Ludi program. ^(b)BS, buried surface: percentage of molecular surface in contact with protein calculated by Ludi based on docked positions. Based on published crystal structures of RT complexes, our calculation shows that these values could be as low as 77%(in RT-HEPT complex) and can be high as 90%(in RT-APA complex) but most of them average around 84%. ^(c)Ludi K_(i)values were calculate based on modified empirical score function in the Ludi program as described for Example 1. (Bohm, J. Comput. Aided. Mol. Des., 1994, 8, 243-256; 1996,) Ideal hydrogen bond distances and angles between compounds and protein are assumed in all cases for Ludi Score and K_(i)calculation. In published crystal structures of RT complexes, hydrogen bond geometries are indeed close to ideal; the amide carbonyl of residue K101 on a loop demonstrates # susbstantial flexibility which can accommodate the best geometry for hydrogen bonding. The number of rotatable bonds (2, or 2 + n for n methoxy groups) is used in the Ludi calculation to reflect loss of binding energy due to freezing of internal degrees of freedom. ^(d)SI (selectivity index) = IC₅₀[MTA]/IC₅₀[p24]). IC₅₀[MTA]values were >100 μM for compounds II-1-9, as well as trovirdine. IC₅₀[MTA]for AZT was 50 μM. N.D., not determined, for compounds with IC₅₀[rRT]greater than 1.0 μM. N.A., not applicable.

Example 8 In virto Assays of PETT Derivatives II

Methoxy Substitutions

The estimated K_(i) values accurately predicted the trend of the measured IC₅₀[rRT] values for the inhibition of recombinant HIV RT. Compound II-4 had the lowest K_(i) value. The docking results showed that the meta-methoxy group of II-4 is situated near Pro95 and Trp229 in the binding site, providing contact with these protein residues which cannot be achieved by trovirdine (FIG. 5). Based on the IC₅₀ [rRT] values of all methoxy compounds, the meta-methoxy substituted compound II-4, which had a K_(i) value of 0.4 μM, showed greater inhibitory activity against recombinant HIV RT and it was approximately 2-fold more potent than trovirdine (IC₅₀[rRT] was 0.4 μM for compound II-4 versus 0.8 μM for trovirdine). Compound II-4 abrogated HIV replication in human peripheral blood mononuclear cells at nanomolar concentrations with an IC₅₀ value of 3 nM and a selectivity index (SI) of >3×10⁴ (Table 6).

Fluorine Substitutions

Among the fluorine (F) substituted compounds II-2, II-5, and II-8, both meta and ortho fluoro compounds were at least 7-fold more active than trovirdine (IC₅₀[p24]<1 nM) (Table 6). Based on the IC₅₀[rRT] values, compounds with F substitutions at the meta and ortho positions had nearly the same inhibitory activity against recombinant HIV RT but the para-F substituted compound was 10-fold less active. The color-coded composite binding pocket (FIG. 5) also shows that Wing 2 is mostly hydrophobic except for the region near the ortho positions on both sides of the phenyl ring where polar groups such as halogen atoms would be compatible. Trovirdine, however, lacks such ring substitutents which could provide favorable interactions with these regions of the binding site based on our modeling. Substitutions at the meta position could be on the polar region or the hydrophobic region depending on the chemical group and its consequent conformational change (FIG. 5). The m-F substituent of compound II-5 is probably exposed to the polar (blue) region and therefore is as active as the o-F group which would also be exposed to the polar region according to our modeling. The trend in IC₅₀[rRT] values observed for F-substituted compounds may reflect such a preference. The p-F atom, which is small in size but electronegative, may not be compatible with the location of the ring plane of nearby hydrophobic Trp229 and could contribute to the lower activity. We postulate that this same incompatibility should be observed for any other highly hydrophilic group at the para position, and that an additional binding penalty be imposed to better quantitate such features when undertaking modeling studies.

Chlorine Substitutions

Chlorine (Cl) substituted compounds II-3, II-6, and II-9 show a trend of observed biological activities which differs from that of both the fluorine and methoxy compounds. Like the p-F substituted compound which was less active than other F-substituted compounds, the p-Cl compound was less active than the o-Cl compound based on the IC₅₀[rRT] values. Unlike the m-F substituted compound which was as active as the o-F substituted compound, the m-Cl compound was not as active as the o-Cl substituted compound. According to our modeling, o-Cl is the most likely substituent to be situated near a limited polar region at Wing 2, an interaction which would be favorable. The o-Cl compound, like the o-F compound discussed above, was in fact more active than trovirdine, as was predicted by the modeling procedure and by the use of the composite binding pocket.

Hydrophobic Group Preferred at the Para Position

When IC₅₀[rRT]values of all compounds with para substitutions are compared (II-7-9), a distinct trend is evident: the p-methoxy (OMe) compound (7) is favored over the p-halogen group compounds (II-8 and II-9) (Table 6). Only the p-OMe substituted PETT derivative, compound II-7, is comparable to trovirdine in its inhibitory activity against recombinant HIV RT. Compound II-7 inhibited HIV replication in peripheral blood mononuclear cells with an IC₅₀ value of 15 nM (Table 6). This p-OMe preference is consistent with the understanding of the color-coded composite binding pocket at Wing 2, where the binding pocket residues near the para position are relatively hydrophobic. One can reasonably assume, based on chemical intuition and the available inhibition data which is consistent with the modeling, that para substituted hydrophobic groups positioned near a hydrophobic region of the pocket are most preferred, followed by halogens, and finally hydrophilic groups.

Conclusions

In summary, the data revealed the following structure-activity relationships affecting the potency of PETT derivatives with substitutions on various positions of the phenyl ring:

1) methoxy substitution is more favorable at the meta position than at the ortho or para positions;

2) fluorine substitution is favorable at ortho and meta positions but not at the para position;

3) chlorine substitution is favorable only at the ortho position;

4) a hydrophobic group is more desirable than a polar group or hydrophilic group at the para position. These results were generally consistent with predictions made during modeling.

The use of the composite NNI binding pocket allowed the identification and structure-based design of at least 3 promising PETT derivatives with ortho-F (II-2), ortho-Cl (II-3), and meta-F (II-5) substituents on the phenyl ring. These novel PETT derivatives were more active than trovirdine (as predicted) or AZT and showed potent anti-HIV activity with IC₅₀[p24] values <1 nM and selectivity indices (SI) of >100,000 (Table 6).

Example 9 Design of Heterocyclic PETT Derivatives III

In the course of the search for potent NNIs, a computer model has been developed in which a composite binding pocket was constructed from nine individual crystal structures of RT-NNI complexes. Modeling studies lead to the identification of a number of NNIs with IC₅₀ values beyond 1 nM for the inhibition of HIV replication (measured by p24 production in HIV-infected human peripheral blood mononuclear cells) and showed no detectable cytotoxicity against human T-lymphocytes (inhibition of cellular proliferation was >100 μM as measured by MTA).

The detailed analysis of trovirdine, a potent PETT derivative, revealed multiple sites which can be used for the incorporation of larger functional groups. In the composite binding pocket, the docked trovirdine molecule showed a lot of usable space surrounding the pyridyl ring, the ethyl linker and near the 5-bromo position (shown in structure of PETT derivative). It was proposed that efficient use of this space by strategically designed functional groups would lead to high affinity binding and ultimately result in better inhibitors.

The effect of systematic substitutions of the phenyl ring of trovirdine with various heterocyclic rings was studied. This provides an alternative strategy to fit the compound into the relatively flexible and spacious Wing 2 region (as illustrated by the composite binding pocket). In the subsequent modeling studies these heterocyclic rings, which have a larger volume than the pyridyl ring of trovirdine, were shown to better fill the Wing 2 region of the composite binding pocket.

The piperidinyl, piperzinyl and morpholinyl rings of compounds III-1-3 are puckered and therefore occupy a larger overall volume than the planar pyridyl ring of trovirdine and are in close contact with residues Leu234 and Leu100, the latter of which can mutate to isoleucine, frequently found in a drug-resistant RT mutant strain. The encouraging results from efforts to make modifications perpendicular to the ring plane introduces new possibilities to develop more potent inhibitors of RT.

The heterocyclic rings which are conformationally more flexible than an aromatic ring may have the advantage of fitting an uncompromising binding pocket more effectively, despite the expense paid for loss of entropy upon binding. Various combinations of double substitutions at axial or equatorial positions of these heterocyclic rings would generate derivatives with a broader range of curvatures than trovirdine derivatives and would serve to better fit Wing 2 which itself contains some curvature.

Example 10 In virto Assays of Anti-HIV-1 Activity Using PETT Derivatives III

Compounds III-1 to III-3 were tested for anti-HIV activity in HTLVIIIB-infected peripheral blood mononuclear cells. Anti-HIV activity was tested using the p24 immunoassay described above for Example 6. Cytotoxicity was also analyzed using a Microculture tetrazolium Assay (MTA), as described above for Example 6.

The data below in Table 7 show the inhibitory effects of PETT derivatives (compounds III-1-3) on p24 production in HIV-infected peripheral blood mononuclear cells and on viability of peripheral blood mononuclear cells. IC₅₀ values represent the concentration required to inhibit by 50% the activity of HIV replication as measured by assays of p24 production (IC₅₀ [p24]) or the concentration required to decrease cellular proliferation by 50% as measured by MTA (IC₅₀ [MTA]) (Uckun, et al., Antimicrobial Agents and Chemotherapy, 1998, 42, 383; Zarling, et al., Nature, 1990, 347, 92-95; Erice, et al., Antimicrob. Ag. Chemother., 1993, 37, 835)

All three compounds III-1-3 were more potent than trovirdine for inhibitition of HIV. Our lead heterocyclic PETT derivatives, N-[2-(1-piperidinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (compound III-1) and N-[2-(1-morpholinylethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (compound 3) elicited potent anti-HIV activity with IC₅₀ values less than 1 nM for the inhibition of HIV replication (measured by p24 production in HIV-infected human peripheral blood mononuclear cells) and showed no detectable cytotoxicity (inhibition of cellular proliferation was >100 μM as measured by MTA) (Table 7).

TABLE 7

IC₅₀[p24] IC₅₀[MTA] Compound R (μM) (μM) SI III-1 piperdinyl <0.001 >100 >1 × 10⁵ III-2 piperazinyl 0.002 >100 >5 × 10⁴ III-3 morpholinyl <0.001 >100 >1 × 10⁵ Trovirdine pyridyl 0.007 >100 >1 × 10⁴ AZT — 0.004 50 7 × 10³

Example 11 “SeeGap” Program for Analysis of Gap Space

To analyze the gap space between the binding pocket and complexed NNI, the “SeeGap” program was developed. The following instructions are for use of the program, whose code is listed below in Table 8:

Preparation

1. Extract the source codes at the lines indicated. The first program is a C-shell command file and should be named as “SeeGap”; the second program should be named as “pdbmax.f”; the third “gridbox.f” and fourth “chgcolor.f”.

2. Compile the source codes: for the first, chmod +x SeeGap; the second, third, and fourth by “f77-o file file.f”.

3. You should now have the executive versions of the programs named as “SeeGap”, “pdbmax”, “gridbox” and “chgcolor”. The preparation is ready.

Use the Program

1. Open “insightII” window, and read in the coordinates of the protein and the coordinates of the ligand. Next, assign the potential to both coordinates by builder module within “insightII” (see insight II manual).

2. Position the ligand in the binding site by a docking procedure, if the position of the ligand is unknown.

3. Using subset/interface command, determine the coordinates of the protein that immediately surround the ligand by a defined distance, e.g., 7 angstroms. Write out the coordinates and name it as “bind.pdb”; write out the coordinates of the ligand and name it as “ligand.pdb”.

4. Adjust the input parameters in the command file “SeeGap” as appropriate.

5. Run the program by typing “SeeGap ligand.pdb bind.pdb>out&”.

6. The results should be in three files: contact.pdb, which represents the grid points on the surface of the ligand and in contact with the protein residues; gap.pdb, which represents the grid points available for modification; and lig.pdb, which represents the grid points covering the ligand.

7. Use a molecular graphics software to display these coordinates.

Table 9: Coordinates of Composite Binding Pocket

These coordinates can be entered into a molecular graphics program to generate a molecular surface representation of the composite binding pocket, which then can be used to design and evaluate inhibitors of RT.

ATOM   1 O H2O   1 144.048 −24.778 68.464 inf inf ATOM   2 O H2O   2 144.416 −24.592 68.433 inf inf ATOM   3 O H2O   3 144.416 −24.225 68.423 inf inf ATOM   4 O H2O   4 143.694 −25.486 68.876 inf inf ATOM   5 O H2O   5 144.048 −25.306 68.683 inf inf ATOM   6 O H2O   6 144.749 −25.257 68.756 inf inf ATOM   7 O H2O   7 143.349 −24.944 68.703 inf inf ATOM   8 O H2O   8 144.790 −24.969 68.630 inf inf ATOM   9 O H2O   9 143.080 −24.603 68.775 inf inf ATOM  10 O H2O  10 145.130 −24.581 68.682 inf inf ATOM  11 O H2O  11 143.639 −24.225 68.487 inf inf ATOM  12 O H2O  12 145.513 −24.404 68.846 inf inf ATOM  13 O H2O  13 143.655 −23.832 68.549 inf inf ATOM  14 O H2O  14 145.157 −23.856 68.637 inf inf ATOM  15 O H2O  15 143.471 −23.455 68.774 inf inf ATOM  16 O H2O  16 144.786 −23.480 68.619 inf inf ATOM  17 O H2O  17 143.670 −23.285 68.803 inf inf ATOM  18 O H2O  18 144.785 −23.149 68.737 inf inf ATOM  19 O H2O  19 144.417 −22.949 68.853 inf inf ATOM  20 O H2O  20 143.693 −25.667 69.048 inf inf ATOM  21 O H2O  21 144.417 −25.702 69.012 inf inf ATOM  22 O H2O  22 143.280 −25.554 69.161 inf inf ATOM  23 O H2O  23 145.154 −25.515 69.200 inf inf ATOM  24 O H2O  24 142.936 −24.965 69.009 inf inf ATOM  25 O H2O  25 142.683 −24.618 69.149 inf inf ATOM  26 O H2O  26 142.673 −24.225 69.139 inf inf ATOM  27 O H2O  27 146.037 −24.225 69.239 inf inf ATOM  28 O H2O  28 146.042 −23.856 69.233 inf inf ATOM  29 O H2O  29 145.586 −23.456 68.921 inf inf ATOM  30 O H2O  30 143.152 −23.144 69.225 inf inf ATOM  31 O H2O  31 145.515 −23.125 69.025 inf inf ATOM  32 O H2O  32 143.661 −22.890 69.155 inf inf ATOM  33 O H2O  33 144.786 −22.742 69.007 inf inf ATOM  34 O H2O  34 144.063 −22.602 69.236 inf inf ATOM  35 O H2O  35 144.048 −26.097 69.620 inf inf ATOM  36 O H2O  36 144.417 −25.997 69.413 inf inf ATOM  37 O H2O  37 143.287 −25.730 69.365 inf inf ATOM  38 O H2O  38 145.148 −25.868 69.584 inf inf ATOM  39 O H2O  39 142.892 −25.364 69.350 inf inf ATOM  40 O H2O  40 142.606 −25.130 69.584 inf inf ATOM  41 O H2O  41 145.857 −25.125 69.596 inf inf ATOM  42 O H2O  42 145.964 −24.629 69.323 inf inf ATOM  43 O H2O  43 146.208 −24.258 69.503 inf inf ATOM  44 O H2O  44 142.554 −23.662 69.558 inf inf ATOM  45 O H2O  45 142.828 −23.175 69.610 inf inf ATOM  46 O H2O  46 143.260 −22.858 69.517 inf inf ATOM  47 O H2O  47 145.718 −22.739 69.559 inf inf ATOM  48 O H2O  48 143.886 −22.425 69.590 inf inf ATOM  49 O H2O  49 144.975 −22.345 69.548 inf inf ATOM  50 O H2O  50 144.786 −22.277 69.595 inf inf ATOM  51 O H2O  51 144.048 −26.251 69.938 inf inf ATOM  52 O H2O  52 144.994 −26.125 69.920 inf inf ATOM  53 O H2O  53 145.525 −25.701 69.751 inf inf ATOM  54 O H2O  54 142.858 −25.603 69.941 inf inf ATOM  55 O H2O  55 142.410 −24.956 69.939 inf inf ATOM  56 O H2O  56 146.247 −24.586 69.759 inf inf ATOM  57 O H2O  57 146.322 −24.242 69.726 inf inf ATOM  58 O H2O  58 146.447 −23.856 69.936 inf inf ATOM  59 O H2O  59 146.368 −23.509 69.971 inf inf ATOM  60 O H2O  60 146.277 −23.296 69.932 inf inf ATOM  61 O H2O  61 145.876 −22.762 69.762 inf inf ATOM  62 O H2O  62 143,833 −22.310 69.916 inf inf ATOM  63 O H2O  63 145.829 −22.628 69.962 inf inf ATOM  64 O H2O  64 145.143 −22.230 69.948 inf inf ATOM  65 O H2O  65 144.048 −26.591 70.339 inf inf ATOM  66 O H2O  66 144.605 −26.461 70.287 inf inf ATOM  67 O H2O  67 144.849 −26.350 70.242 inf inf ATOM  68 O H2O  68 143.010 −25.838 70.326 inf inf ATOM  69 O H2O  69 145.844 −25.653 70.169 inf inf ATOM  70 O H2O  70 142.505 −25.253 70.305 inf inf ATOM  71 O H2O  71 146.408 −25.313 70.366 inf inf ATOM  72 O H2O  72 142.287 −24.619 70.305 inf inf ATOM  73 O H2O  73 142.270 −24.225 70.305 inf inf ATOM  74 O H2O  74 146.581 −23.856 70.155 inf inf ATOM  75 O H2O  75 146.640 −23.667 70.298 inf inf ATOM  76 O H2O  76 146.387 −23.165 70.341 inf inf ATOM  77 O H2O  77 146.235 −22.946 70.319 inf inf ATOM  78 O H2O  78 145.533 −22.364 70.118 inf inf ATOM  79 O H2O  79 144.038 −22.156 70.305 inf inf ATOM  80 O H2O  80 145.471 −22.274 70.333 inf inf ATOM  81 O H2O  81 144.048 −27.016 70.623 inf inf ATOM  82 O H2O  82 144.634 −26.841 70.626 inf inf ATOM  83 O H2O  83 144.819 −26.507 70.435 inf inf ATOM  84 O H2O  84 145.332 −26.427 70.685 inf inf ATOM  85 O H2O  85 145.880 −26.228 70.717 inf inf ATOM  86 O H2O  86 142.907 −25.909 70.653 inf inf ATOM  87 O H2O  87 146.588 −25.657 70.623 inf inf ATOM  88 O H2O  88 147.374 −25.700 70.660 inf inf ATOM  89 O H2O  89 148.108 −25.686 70.594 inf inf ATOM  90 O H2O  90 142.531 −25.283 70.673 inf inf ATOM  91 O H2O  91 147.001 −25.530 70.644 inf inf ATOM  92 O H2O  92 148.427 −25.333 70.643 inf inf ATOM  93 O H2O  93 146.982 −24.943 70.558 inf inf ATOM  94 O H2O  94 148.109 −25.140 70.625 inf inf ATOM  95 O H2O  95 147.195 −24.587 70.651 inf inf ATOM  96 O H2O  96 147.177 −24.225 70.696 inf inf ATOM  97 O H2O  97 142.471 −23.515 70.647 inf inf ATOM  98 O H2O  98 142.595 −23.318 70.666 inf inf ATOM  99 O H2O  99 142.934 −22.926 70.677 inf inf ATOM  100 O H2O  100 146.583 −22.969 70.735 inf inf ATOM  101 O H2O  101 146.022 −22.436 70.730 inf inf ATOM  102 O H2O  102 144.417 −22.087 70.674 inf inf ATOM  103 O H2O  103 145.844 −22.277 70.742 inf inf ATOM  104 O H2O  104 144.233 −27.553 71.039 inf inf ATOM  105 O H2O  105 143.655 −27.432 70.974 inf inf ATOM  106 O H2O  106 144.442 −27.438 70.968 inf inf ATOM  107 O H2O  107 142.971 −26.975 71.068 inf inf ATOM  108 O H2O  108 144.850 −26.872 70.763 inf inf ATOM  109 O H2O  109 142.790 −26.440 71.066 inf inf ATOM  110 O H2O  110 145.888 −26.614 71.059 inf inf ATOM  111 O H2O  111 147.185 −26.441 71.041 inf inf ATOM  112 O H2O  112 148.109 −26.648 71.020 inf inf ATOM  113 O H2O  113 148.669 −26.449 71.032 inf inf ATOM  114 O H2O  114 146.285 −26.324 70.974 inf inf ATOM  115 O H2O  115 147.001 −26.084 70.828 inf inf ATOM  116 O H2O  116 148.503 −26.108 70.772 inf inf ATOM  117 O H2O  117 142.649 −25.772 70.972 inf inf ATOM  118 O H2O  118 142.535 −25.326 71.039 inf inf ATOM  119 O H2O  119 142.463 −24.937 71.041 inf inf ATOM  120 O H2O  120 148.837 −24.973 70.888 inf inf ATOM  121 O H2O  121 147.762 −24.573 70.772 inf inf ATOM  122 O H2O  122 149.033 −24.594 71.039 inf inf ATOM  123 O H2O  123 148.108 −24.225 70.852 inf inf ATOM  124 O H2O  124 142.459 −23.880 71.019 inf inf ATOM  125 O H2O  125 148.477 −23.866 70.928 inf inf ATOM  126 O H2O  126 152.550 −23.661 71.065 inf inf ATOM  127 O H2O  127 147.710 −23.518 70.952 inf inf ATOM  128 O H2O  128 148.845 −23.672 71.048 inf inf ATOM  129 O H2O  129 147.390 −23.272 70.974 inf inf ATOM  130 O H2O  130 143.004 −22.996 71.018 inf inf ATOM  131 O H2O  131 147.021 −22.918 71.009 inf inf ATOM  132 O H2O  132 143.843 −22.331 71.057 inf inf ATOM  133 O H2O  133 144.057 −22.209 71.039 inf inf ATOM  134 O H2O  134 145.155 −22.003 70.856 inf inf ATOM  135 O H2O  135 146.253 −22.218 71.067 inf inf ATOM  136 O H2O  136 145.894 −21.890 71.108 inf inf ATOM  137 O H2O  137 143.673 −27.752 71.404 inf inf ATOM  138 O H2O  138 144.425 −27.759 71.401 inf inf ATOM  139 O H2O  139 142.960 −27.339 71.427 inf inf ATOM  140 O H2O  140 145.148 −27.353 71.418 inf inf ATOM  141 O H2O  141 145.550 −27.062 71.366 inf inf ATOM  142 O H2O  142 146.233 −26.749 71.320 inf inf ATOM  143 O H2O  143 147.403 −26.893 71.480 inf inf ATOM  144 O H2O  144 147.735 −26.822 71.219 inf inf ATOM  145 O H2O  145 148.468 −26.781 71.247 inf inf ATOM  146 O H2O  146 142.643 −26.440 71.276 inf inf ATOM  147 O H2O  147 147.003 −26.730 71.337 inf inf ATOM  148 O H2O  148 142.446 −26.051 71.452 inf inf ATOM  149 O H2O  149 149.369 −26.060 71.434 inf inf ATOM  150 O H2O  150 149.447 −25.719 71.367 inf inf ATOM  151 O H2O  151 142.424 −24.951 71.422 inf inf ATOM  152 O H2O  152 149.685 −24.933 71.469 inf inf ATOM  153 O H2O  153 149.734 −24.594 71.488 inf inf ATOM  154 O H2O  154 149.268 −24.225 71.124 inf inf ATOM  155 O H2O  155 142.731 −23.841 71.448 inf inf ATOM  156 O H2O  156 142.812 −23.520 71.368 inf inf ATOM  157 O H2O  157 149.748 −23.478 71.434 inf inf ATOM  158 O H2O  158 147.423 −23.050 71.114 inf inf ATOM  159 O H2O  159 148.867 −23.117 71.149 inf inf ATOM  160 O H2O  160 143.329 −22.764 71.216 inf inf ATOM  161 O H2O  161 147.365 −22.759 71.245 inf inf ATOM  162 O H2O  162 148.847 −22.748 71.181 inf inf ATOM  163 O H2O  163 143.692 −22.585 71.396 inf inf ATOM  164 O H2O  164 147.183 −22.382 71.418 inf inf ATOM  165 O H2O  165 148.288 −22.374 71.403 inf inf ATOM  166 O H2O  166 149.548 −22.416 71.339 inf inf ATOM  167 O H2O  167 144.299 −22.076 71.413 inf inf ATOM  168 O H2O  168 146.991 −22.215 71.443 inf inf ATOM  169 O H2O  169 149.576 −22.203 71.431 inf inf ATOM  170 O H2O  170 145.001 −21.694 71.453 inf inf ATOM  171 O H2O  171 146.443 −21.649 71.443 inf inf ATOM  172 O H2O  172 145.894 −21.452 71.403 inf inf ATOM  173 O H2O  173 143.692 −27.877 71.623 inf inf ATOM  174 O H2O  174 144.406 −27.883 71.619 inf inf ATOM  175 O H2O  175 144.818 −27.779 71.750 inf inf ATOM  176 O H2O  176 142.717 −27.204 71.769 inf inf ATOM  177 O H2O  177 145.720 −27.202 71.768 inf inf ATOM  178 O H2O  178 145.935 −27.086 71.728 inf inf ATOM  179 O H2O  179 147.001 −26.972 71.791 inf inf ATOM  180 O H2O  180 148.495 −27.054 71.756 inf inf ATOM  181 O H2O  181 142.384 −26.441 71.781 inf inf ATOM  182 O H2O  182 142.297 −26.096 71.743 inf inf ATOM  183 O H2O  183 149.650 −25.913 71.745 inf inf ATOM  184 O H2O  184 149.804 −25.332 71.759 inf inf ATOM  185 O H2O  185 142.522 −24.758 71.783 inf inf ATOM  186 O H2O  186 150.044 −24.594 71.845 inf inf ATOM  187 O H2O  187 142.920 −23.846 71.616 inf inf ATOM  188 O H2O  188 150.094 −23.840 71.811 inf inf ATOM  189 O H2O  189 150.140 −23.487 71.781 inf inf ATOM  190 O H2O  190 149.996 −23.128 71.524 inf inf ATOM  191 O H2O  191 143.870 −22.754 71.775 inf inf ATOM  192 O H2O  192 144.036 −22.552 71.787 inf inf ATOM  193 O H2O  193 148.080 −22.338 71.501 inf inf ATOM  194 O H2O  194 147.010 −21.997 71.566 inf inf ATOM  195 O H2O  195 148.458 −21.971 71.538 inf inf ATOM  196 O H2O  196 149.817 −21.962 71.710 inf inf ATOM  197 O H2O  197 144.643 −21.670 71.794 inf inf ATOM  198 O H2O  198 147.377 −21.815 71.758 inf inf ATOM  199 O H2O  199 148.660 −21.627 71.771 inf inf ATOM  200 O H2O  200 149.604 −21.778 71.734 inf inf ATOM  201 O H2O  201 145.510 −21.250 71.547 inf inf ATOM  202 O H2O  202 146.868 −21.251 71.710 inf inf ATOM  203 O H2O  203 145.161 −21.090 71.791 inf inf ATOM  204 O H2O  204 146.261 −20.905 71.603 inf inf ATOM  205 O H2O  205 145.710 −20.536 71.791 inf inf ATOM  206 O H2O  206 146.621 −20.740 71.815 inf inf ATOM  207 O H2O  207 143.707 −28.248 72.013 inf inf ATOM  208 O H2O  208 144.405 −28.256 71.996 inf inf ATOM  209 O H2O  209 143.294 −27.935 71.947 inf inf ATOM  210 O H2O  210 142.946 −27.729 72.153 inf inf ATOM  211 O H2O  211 145.390 −27.597 72.111 inf inf ATOM  212 O H2O  212 145.884 −27.333 72.171 inf inf ATOM  213 O H2O  213 147.742 −27.170 71.967 inf inf ATOM  214 O H2O  214 142.440 −26.773 72.151 inf inf ATOM  215 O H2O  215 147.002 −27.056 72.135 inf inf ATOM  216 O H2O  216 149.074 −26.861 72.124 inf inf ATOM  217 O H2O  217 149.521 −26.560 72.216 inf inf ATOM  218 O H2O  218 142.208 −26.069 71.967 inf inf ATOM  219 O H2O  219 142.199 −25.701 71.966 inf inf ATOM  220 O H2O  220 142.187 −25.515 72.147 inf inf ATOM  221 O H2O  221 142.397 −24.970 72.151 inf inf ATOM  222 O H2O  222 142.720 −24.572 72.153 inf inf ATOM  223 O H2O  223 143.061 −24.180 72.171 inf inf ATOM  224 O H2O  224 143.358 −23.534 71.918 inf inf ATOM  225 O H2O  225 150.315 −23.667 72.155 inf inf ATOM  226 O H2O  226 143.910 −23.165 72.103 inf inf ATOM  227 O H2O  227 144.088 −22.957 72.119 inf inf ATOM  228 O H2O  228 144.267 −22.388 72.138 inf inf ATOM  229 O H2O  229 144.380 −22.178 72.162 inf inf ATOM  230 O H2O  230 150.348 −22.182 72.139 inf inf ATOM  231 O H2O  231 148.108 −21.617 71.951 inf inf ATOM  232 O H2O  232 150.013 −21.767 72.104 inf inf ATOM  233 O H2O  233 147.340 −21.307 72.000 inf inf ATOM  234 O H2O  234 148.473 −21.440 72.140 inf inf ATOM  235 O H2O  235 144.704 −20.904 72.218 inf inf ATOM  236 O H2O  236 147.177 −20.908 72.158 inf inf ATOM  237 O H2O  237 145.147 −20.533 71.955 inf inf ATOM  238 O H2O  238 146.825 −20.525 72.144 inf inf ATOM  239 O H2O  239 148.833 −20.164 72.106 inf inf ATOM  240 O H2O  240 146.241 −20.189 72.032 inf inf ATOM  241 O H2O  241 144.952 −19.783 72.107 inf inf ATOM  242 O H2O  242 146.216 −19.842 72.107 inf inf ATOM  243 O H2O  243 145.525 −19.468 72.091 inf inf ATOM  244 O H2O  244 145.524 −19.285 72.215 inf inf ATOM  245 O H2O  245 144.048 −28.821 72.532 inf inf ATOM  246 O H2O  246 144.620 −28.691 72.489 inf inf ATOM  247 O H2O  247 144.840 −28.339 72.255 inf inf ATOM  248 O H2O  248 145.273 −28.245 72.573 inf inf ATOM  249 O H2O  249 145.206 −27.957 72.285 inf inf ATOM  250 O H2O  250 145.561 −27.779 72.473 inf inf ATOM  251 O H2O  251 142.595 −27.218 72.480 inf inf ATOM  252 O H2O  252 146.633 −27.181 72.334 inf inf ATOM  253 O H2O  253 147.370 −27.155 72.339 inf inf ATOM  254 O H2O  254 142.416 −26.796 72.520 inf inf ATOM  255 O H2O  255 149.241 −26.847 72.309 inf inf ATOM  256 O H2O  256 149.756 −26.795 72.547 inf inf ATOM  257 O H2O  257 150.146 −26.445 72.502 inf inf ATOM  258 O H2O  258 150.259 −26.038 72.429 inf inf ATOM  259 O H2O  259 150.293 −25.686 72.382 inf inf ATOM  260 O H2O  260 150.311 −25.332 72.353 inf inf ATOM  261 O H2O  261 150.496 −24.963 72.533 inf inf ATOM  262 O H2O  262 150.406 −24.631 72.557 inf inf ATOM  263 O H2O  263 150.332 −24.408 72.517 inf inf ATOM  264 O H2O  264 150.307 −23.307 72.338 inf inf ATOM  265 O H2O  265 143.671 −23.664 72.523 inf inf ATOM  266 O H2O  266 144.054 −23.308 72.517 inf inf ATOM  267 O H2O  267 150.636 −22.748 72.488 inf inf ATOM  268 O H2O  268 150.564 −22.365 72.506 inf inf ATOM  269 O H2O  269 144.546 −21.640 72.520 inf inf ATOM  270 O H2O  270 144.506 −21.295 72.521 inf inf ATOM  271 O H2O  271 148.847 −21.270 72.335 inf inf ATOM  272 O H2O  272 149.923 −21.503 72.536 inf inf ATOM  273 O H2O  273 147.750 −21.063 72.513 inf inf ATOM  274 O H2O  274 149.215 −21.213 72.561 inf inf ATOM  275 O H2O  275 144.701 −20.533 72.216 inf inf ATOM  276 O H2O  276 144.291 −20.164 72.559 inf inf ATOM  277 O H2O  277 147.001 −20.349 72.520 inf inf ATOM  278 O H2O  278 146.596 −19.809 72.371 inf inf ATOM  279 O H2O  279 144.782 −19.424 82.329 inf inf ATOM  280 O H2O  280 146.486 −19.395 72.481 inf inf ATOM  281 O H2O  281 145.159 −19.062 72.347 inf inf ATOM  282 O H2O  282 146.294 −19.210 72.473 inf inf ATOM  283 O H2O  283 145.525 −18.765 72.451 inf inf ATOM  284 O H2O  284 145.524 −18.548 72.587 inf inf ATOM  285 O H2O  285 143.655 −28.924 72.853 inf inf ATOM  286 O H2O  286 144.789 −28.850 72.884 inf inf ATOM  287 O H2O  287 142.895 −28.315 72.675 inf inf ATOM  288 O H2O  288 145.572 −27.954 72.657 inf inf ATOM  289 O H2O  289 142.485 −27.547 72.922 inf inf ATOM  290 O H2O  290 146.244 −27.683 72.938 inf inf ATOM  291 O H2O  291 146.672 −27.456 72.863 inf inf ATOM  292 O H2O  292 147.551 −27.151 72.889 inf inf ATOM  293 O H2O  293 148.476 −27.172 72.705 inf inf ATOM  294 O H2O  294 149.218 −27.105 72.738 inf inf ATOM  295 O H2O  295 148.109 −27.148 72.891 inf inf ATOM  296 O H2O  296 149.954 −27.067 72.816 inf inf ATOM  297 O H2O  297 142.307 −26.459 72.889 inf inf ATOM  298 O H2O  298 150.882 −26.444 72.884 inf inf ATOM  299 O H2O  299 151.038 −26.047 72.773 inf inf ATOM  300 O H2O  300 142.238 −25.701 72.926 inf inf ATOM  301 O H2O  301 142.319 −25.313 72.918 inf inf ATOM  302 O H2O  302 142.449 −25.005 72.868 inf inf ATOM  303 O H2O  303 142.596 −24.804 72.879 inf inf ATOM  304 O H2O  304 142.983 −24.473 72.868 inf inf ATOM  305 O H2O  305 150.375 −24.223 72.681 inf inf ATOM  306 O H2O  306 143.829 −23.829 72.923 inf inf ATOM  307 O H2O  307 144.187 −23.457 72.919 inf inf ATOM  308 O H2O  308 144.433 −22.753 72.702 inf inf ATOM  309 O H2O  309 144.507 −22.378 72.891 inf inf ATOM  310 O H2O  310 144.506 −21.640 72.865 inf inf ATOM  311 O H2O  311 149.407 −21.230 72.889 inf inf ATOM  312 O H2O  312 144.269 −20.878 72.901 inf inf ATOM  313 O H2O  313 148.093 −21.141 72.906 inf inf ATOM  314 O H2O  314 144.164 −20.553 72.860 inf inf ATOM  315 O H2O  315 147.019 −20.158 72.693 inf inf ATOM  316 O H2O  316 147.029 −19.967 72.783 inf inf ATOM  317 O H2O  317 146.823 −19.421 72.885 inf inf ATOM  318 O H2O  318 146.568 −19.105 72.785 inf inf ATOM  319 O H2O  319 144.769 −18.654 72.654 inf inf ATOM  320 O H2O  320 146.585 −18.900 72.927 inf inf ATOM  321 O H2O  321 144.967 −18.297 72.871 inf inf ATOM  322 O H2O  322 146.251 −18.515 72.901 inf inf ATOM  323 O H2O  323 143.679 −29.093 73.301 inf inf ATOM  324 O H2O  324 144.048 −29.055 83.069 inf inf ATOM  325 O H2O  325 144.770 −28.976 73.106 inf inf ATOM  326 O H2O  326 142.958 −28.633 73.087 inf inf ATOM  327 O H2O  327 145.380 −28.694 73.227 inf inf ATOM  328 O H2O  328 145.577 −28.329 73.048 inf inf ATOM  329 O H2O  329 142.521 −27.931 73.052 inf inf ATOM  330 O H2O  330 142.378 −27.547 73.255 inf inf ATOM  331 O H2O  331 146.704 −27.506 73.258 inf inf ATOM  332 O H2O  332 148.291 −27.190 73.258 inf inf ATOM  333 O H2O  333 149.222 −27.345 73.264 inf inf ATOM  334 O H2O  334 149.954 −27.252 72.999 inf inf ATOM  335 O H2O  335 142.337 −26.809 73.259 inf inf ATOM  336 O H2O  336 150.742 −26.848 73.025 inf inf ATOM  337 O H2O  337 151.074 −26.450 73.061 inf inf ATOM  338 O H2O  338 151.404 −26.061 73.092 inf inf ATOM  339 O H2O  339 151.452 −25.701 73.059 inf inf ATOM  340 O H2O  340 151.507 −25.368 73.295 inf inf ATOM  341 O H2O  341 151.051 −24.974 73.074 inf inf ATOM  342 O H2O  342 142.913 −24.761 73.277 inf inf ATOM  343 O H2O  343 151.019 −24.821 73.275 inf inf ATOM  344 O H2O  344 143.838 −24.201 73.278 inf inf ATOM  345 O H2O  345 144.025 −24.018 73.276 inf inf ATOM  346 O H2O  346 150.577 −23.486 73.276 inf inf ATOM  347 O H2O  347 150.615 −23.144 73.285 inf inf ATOM  348 O H2O  348 150.557 −22.367 73.271 inf inf ATOM  349 O H2O  349 150.114 −21.665 73.249 inf inf ATOM  350 O H2O  350 144.393 −21.278 73.063 inf inf ATOM  351 O H2O  351 144.186 −20.933 73.243 inf inf ATOM  352 O H2O  352 148.455 −21.162 73.234 inf inf ATOM  353 O H2O  353 143.997 −20.489 73.302 inf inf ATOM  354 O H2O  354 147.700 −20.766 73.287 inf inf ATOM  355 O H2O  355 147.358 −20.355 73.264 inf inf ATOM  356 O H2O  356 147.111 −19.822 73.284 inf inf ATOM  357 O H2O  357 147.031 −19.598 73.250 inf inf ATOM  358 O H2O  358 144.017 −18.857 73.243 inf inf ATOM  359 O H2O  359 144.347 −18.433 73.203 inf inf ATOM  360 O H2O  360 146.418 −18.342 73.276 inf inf ATOM  361 O H2O  361 145.524 −18.014 73.198 inf inf ATOM  362 O H2O  362 143.104 −29.065 73.593 inf inf ATOM  363 O H2O  363 144.417 −29.211 73.626 inf inf ATOM  364 O H2O  364 145.333 −29.010 73.634 inf inf ATOM  365 O H2O  365 142.896 −28.894 73.572 inf inf ATOM  366 O H2O  366 142.329 −28.332 73.570 inf inf ATOM  367 O H2O  367 142.209 −28.100 73.633 inf inf ATOM  368 O H2O  368 142.135 −27.587 73.705 inf inf ATOM  369 O H2O  369 146.556 −27.657 73.627 inf inf ATOM  370 O H2O  370 149.585 −27.526 73.452 inf inf ATOM  371 O H2O  371 142.225 −27.336 73.640 inf inf ATOM  372 O H2O  372 147.329 −27.239 73.631 inf inf ATOM  373 O H2O  373 148.110 −27.171 73.444 inf inf ATOM  374 O H2O  374 150.381 −27.477 73.599 inf inf ATOM  375 O H2O  375 142.298 −26.809 73.606 inf inf ATOM  376 O H2O  376 151.190 −26.771 73.646 inf inf ATOM  377 O H2O  377 151.474 −26.274 73.627 inf inf ATOM  378 O H2O  378 142.560 −25.327 73.448 inf inf ATOM  379 O H2O  379 142.899 −24.949 73.464 inf inf ATOM  380 O H2O  380 151.445 −25.138 73.627 inf inf ATOM  381 O H2O  381 143.651 −24.539 73.491 inf inf ATOM  382 O H2O  382 144.023 −24.206 73.468 inf inf ATOM  383 O H2O  383 150.752 −24.370 73.668 inf inf ATOM  384 O H2O  384 144.417 −23.671 73.628 inf inf ATOM  385 O H2O  385 150.501 −23.117 73.625 inf inf ATOM  386 O H2O  386 150.448 −22.399 73.607 inf inf ATOM  387 O H2O  387 150.328 −22.007 73.445 inf inf ATOM  388 O H2O  388 149.971 −21.620 73.455 inf inf ATOM  389 O H2O  389 148.882 −21.317 73.662 inf inf ATOM  390 O H2O  390 149.581 −21.467 73.620 inf inf ATOM  391 O H2O  391 148.436 −21.223 73.625 inf inf ATOM  392 O H2O  392 147.726 −20.728 73.627 inf inf ATOM  393 O H2O  393 143.766 −19.820 73.603 inf inf ATOM  394 O H2O  394 147.031 −19.417 73.430 inf inf ATOM  395 O H2O  395 147.037 −19.236 73.617 inf inf ATOM  396 O H2O  396 144.068 −18.517 73.634 inf inf ATOM  397 O H2O  397 146.682 −18.461 73.619 inf inf ATOM  398 O H2O  398 144.965 −17.912 73.617 inf inf ATOM  399 O H2O  399 146.060 −17.991 73.640 inf inf ATOM  400 O H2O  400 146.632 −33.227 74.059 inf inf ATOM  401 O H2O  401 145.905 −32.888 74.020 inf inf ATOM  402 O H2O  402 146.279 −32.707 73.869 inf inf ATOM  403 O H2O  403 147.184 −32.714 73.999 inf inf ATOM  404 O H2O  404 146.632 −32.346 73.829 inf inf ATOM  405 O H2O  405 146.053 −31.960 73.939 inf inf ATOM  406 O H2O  406 147.180 −31.981 74.010 inf inf ATOM  407 O H2O  407 143.310 −29.504 74.022 inf inf ATOM  408 O H2O  408 143.679 −29.392 73.812 inf inf ATOM  409 O H2O  409 144.417 −29.333 73.852 inf inf ATOM  410 O H2O  410 145.100 −29.337 73.867 inf inf ATOM  411 O H2O  411 142.614 −28.981 73.855 inf inf ATOM  412 O H2O  412 145.487 −29.185 74.026 inf inf ATOM  413 O H2O  413 142.255 −28.613 73.844 inf inf ATOM  414 O H2O  414 145.933 −28.503 73.976 inf inf ATOM  415 O H2O  415 146.257 −28.094 74.002 inf inf ATOM  416 O H2O  416 146.799 −27.521 74.005 inf inf ATOM  417 O H2O  417 149.597 −27.646 73.997 inf inf ATOM  418 O H2O  418 150.489 −27.502 73.997 inf inf ATOM  419 O H2O  419 147.350 −27.300 74.017 inf inf ATOM  420 O H2O  420 147.920 −27.149 73.996 inf inf ATOM  421 O H2O  421 150.704 −27.380 73.997 inf inf ATOM  422 O H2O  422 148.107 −27.138 73.998 inf inf ATOM  423 O H2O  423 151.377 −26.473 73.997 inf inf ATOM  424 O H2O  424 142.586 −25.709 73.804 inf inf ATOM  425 O H2O  425 142.889 −25.291 73.864 inf inf ATOM  426 O H2O  426 143.287 −24.929 73.855 inf inf ATOM  427 O H2O  427 151.393 −25.172 73.984 inf inf ATOM  428 O H2O  428 151.028 −24.829 73.979 inf inf ATOM  429 O H2O  429 150.643 −24.442 73.980 inf inf ATOM  430 O H2O  430 150.377 −23.898 73.952 inf inf ATOM  431 O H2O  431 150.360 −23.487 73.828 inf inf ATOM  432 O H2O  432 144.583 −22.748 73.997 inf inf ATOM  433 O H2O  433 150.148 −22.375 74.006 inf inf ATOM  434 O H2O  434 150.009 −22.167 74.042 inf inf ATOM  435 O H2O  435 149.638 −21.773 74.039 inf inf ATOM  436 O H2O  436 148.639 −21.306 73.985 inf inf ATOM  437 O H2O  437 147.933 −20.892 73.993 inf inf ATOM  438 O H2O  438 144.104 −20.681 73.977 inf inf ATOM  439 O H2O  439 147.510 −20.183 73.997 inf inf ATOM  440 O H2O  440 143.701 −19.426 73.997 inf inf ATOM  441 O H2O  441 147.048 −19.102 73.997 inf inf ATOM  442 O H2O  442 144.040 −18.498 73.997 inf inf ATOM  443 O H2O  443 144.417 −18.134 73.997 inf inf ATOM  444 O H2O  444 146.313 −18.058 73.973 inf inf ATOM  445 O H2O  445 145.525 −17.795 74.005 inf inf ATOM  446 O H2O  446 145.950 −33.378 74.276 inf inf ATOM  447 O H2O  447 146.632 −33.439 74.203 inf inf ATOM  448 O H2O  448 145.564 −33.239 74.415 inf inf ATOM  449 O H2O  449 146.228 −33.152 74.044 inf inf ATOM  450 O H2O  450 147.550 −33.080 74.370 inf inf ATOM  451 O H2O  451 147.629 −32.740 74.292 inf inf ATOM  452 O H2O  452 147.657 −32.346 74.263 inf inf ATOM  453 O H2O  453 145.534 −31.982 74.195 inf inf ATOM  454 O H2O  454 147.742 −32.161 74.363 inf inf ATOM  455 O H2O  455 146.233 −31.549 74.064 inf inf ATOM  456 O H2O  456 147.537 −31.618 74.380 inf inf ATOM  457 O H2O  457 146.263 −31.252 74.213 inf inf ATOM  458 O H2O  458 147.360 −31.433 74.376 inf inf ATOM  459 O H2O  459 146.079 −30.868 74.361 inf inf ATOM  460 O H2O  460 145.894 −30.688 74.389 inf inf ATOM  461 O H2O  461 144.803 −29.908 74.419 inf inf ATOM  462 O H2O  462 145.517 −29.942 74.392 inf inf ATOM  463 O H2O  463 142.742 −29.414 74.359 inf inf ATOM  464 O H2O  464 144.038 −29.641 74.329 inf inf ATOM  465 O H2O  465 142.522 −29.258 74.346 inf inf ATOM  466 O H2O  466 142.029 −28.647 74.369 inf inf ATOM  467 O H2O  467 141.888 −28.318 74.333 inf inf ATOM  468 O H2O  468 141.828 −28.102 74.364 inf inf ATOM  469 O H2O  469 141.799 −27.597 74.416 inf inf ATOM  470 O H2O  470 146.939 −27.655 74.412 inf inf ATOM  471 O H2O  471 149.393 −27.598 74.366 inf inf ATOM  472 O H2O  472 141.868 −27.378 74.376 inf inf ATOM  473 O H2O  473 148.109 −27.149 74.185 inf inf ATOM  474 O H2O  474 149.148 −27.501 74.404 inf inf ATOM  475 O H2O  475 142.122 −26.878 74.401 inf inf ATOM  476 O H2O  476 142.357 −26.423 74.362 inf inf ATOM  477 O H2O  477 151.408 −26.071 74.406 inf inf ATOM  478 O H2O  478 151.467 −25.701 74.197 inf inf ATOM  479 O H2O  479 151.398 −25.349 74.165 inf inf ATOM  480 O H2O  480 151.142 −25.091 74.387 inf inf ATOM  481 O H2O  481 143.987 −24.717 74.415 inf inf ATOM  482 O H2O  482 150.699 −24.589 74.182 inf inf ATOM  483 O H2O  483 150.364 −24.199 74.209 inf inf ATOM  484 O H2O  484 150.263 −23.873 74.140 inf inf ATOM  485 O H2O  485 150.049 −23.117 74.307 inf inf ATOM  486 O H2O  486 149.850 −22.354 74.410 inf inf ATOM  487 O H2O  487 149.741 −22.038 74.354 inf inf ATOM  488 O H2O  488 144.429 −21.268 74.180 inf inf ATOM  489 O H2O  489 148.486 −21.257 74.184 inf inf ATOM  490 O H2O  490 144.383 −21.102 74.366 inf inf ATOM  491 O H2O  491 144.122 −20/597 74.382 inf inf ATOM  492 O H2O  492 143.992 −20.386 74.384 inf inf ATOM  493 O H2O  493 147.400 −19.967 74.366 inf inf ATOM  494 O H2O  494 147.102 −19.084 74.392 inf inf ATOM  495 O H2O  495 144.063 −18.513 74.361 inf inf ATOM  496 O H2O  496 144.606 −17.960 74.369 inf inf ATOM  497 O H2O  497 144.816 −17.851 74.396 inf inf ATOM  498 O H2O  498 146.240 −17.802 74.395 inf inf ATOM  499 O H2O  499 146.631 −33.922 74.791 inf inf ATOM  500 O H2O  500 147.185 −33.821 74.737 inf inf ATOM  501 O H2O  501 145.852 −33.700 74.693 inf inf ATOM  502 O H2O  502 145.259 −33.138 74.681 inf inf ATOM  503 O H2O  503 145.124 −32.910 74.714 inf inf ATOM  504 O H2O  504 147.981 −32.716 74.697 inf inf ATOM  505 O H2O  505 144.951 −31.984 74.721 inf inf ATOM  506 O H2O  506 144.918 −31.629 74.662 inf inf ATOM  507 O H2O  507 144.780 −31.424 74.278 inf inf ATOM  508 O H2O  508 147.361 −31.248 74.560 inf inf ATOM  509 O H2O  509 144.868 −30.868 74.673 inf inf ATOM  510 O H2O  510 146.643 −30.853 74.512 inf inf ATOM  511 O H2O  511 144.618 −30.492 74.760 inf inf ATOM  512 O H2O  512 145.919 −30.476 74.452 inf inf ATOM  513 O H2O  513 146.961 −30.715 74.785 inf inf ATOM  514 O H2O  514 144.787 −30.130 74.553 inf inf ATOM  515 O H2O  515 146.222 −30.173 74.672 inf inf ATOM  516 O H2O  516 143.514 −29.717 74.735 inf inf ATOM  517 O H2O  517 145.875 −29.762 74.579 inf inf ATOM  518 O H2O  518 142.953 −29.549 74.735 inf inf ATOM  519 O H2O  519 142.311 −29.099 74.735 inf inf ATOM  520 O H2O  520 142.149 −28.876 74.735 inf inf ATOM  521 O H2O  521 141.846 −27.328 74.735 inf inf ATOM  522 O H2O  522 146.679 −27.955 74.528 inf inf ATOM  523 O H2O  523 147.020 −27.761 74.716 inf inf ATOM  524 O H2O  524 149.954 −27.692 74.735 inf inf ATOM  525 O H2O  525 141.934 −27.136 74.735 inf inf ATOM  526 O H2O  526 148.475 −27.324 74.735 inf inf ATOM  527 O H2O  527 150.829 −27.130 74.716 inf inf ATOM  528 O H2O  528 142.224 −26.634 74.745 inf inf ATOM  529 O H2O  529 151.295 −26.071 74.756 inf inf ATOM  530 O H2O  530 143.022 −25.598 74.735 inf inf ATOM  531 O H2O  531 151.176 −25.356 74.711 inf inf ATOM  532 O H2O  532 150.884 −24.956 74.740 inf inf ATOM  533 O H2O  533 150.481 −24.647 74.708 inf inf ATOM  534 O H2O  534 144.506 −24.172 74.736 inf inf ATOM  535 O H2O  535 149.803 −23.846 74.749 inf inf ATOM  536 O H2O  536 149.764 −23.486 74.733 inf inf ATOM  537 O H2O  537 149.926 −23.117 74.538 inf inf ATOM  538 O H2O  538 144.566 −22.379 74.735 inf inf ATOM  539 O H2O  539 144.466 −21.675 74.701 inf inf ATOM  540 O H2O  540 144.375 −21.264 74.735 inf inf ATOM  541 O H2O  541 148.644 −21.314 74.746 inf inf ATOM  542 O H2O  542 148.162 −21.002 74.708 inf inf ATOM  543 O H2O  543 147.801 −20.671 74.714 inf inf ATOM  544 O H2O  544 143.884 −19.795 74.726 inf inf ATOM  545 O H2O  545 147.264 −19.407 74.735 inf inf ATOM  546 O H2O  546 147.020 −18.682 74.542 inf inf ATOM  547 O H2O  547 146.925 −18.257 74.689 inf inf ATOM  548 O H2O  548 146.626 −17.957 74.555 inf inf ATOM  549 O H2O  549 144.985 −17.608 74.749 inf inf ATOM  550 O H2O  550 146.094 −17.544 74.714 inf inf ATOM  551 O H2O  551 145.870 −17.469 74.759 inf inf ATOM  552 O H2O  552 147.370 −34.334 75.146 inf inf ATOM  553 O H2O  553 145.716 −33.812 75.107 inf inf ATOM  554 O H2O  554 147.399 −33.881 74.831 inf inf ATOM  555 O H2O  555 148.040 −33.789 75.022 inf inf ATOM  556 O H2O  556 147.806 −33.504 74.803 inf inf ATOM  557 O H2O  557 145.017 −33.068 75.119 inf inf ATOM  558 O H2O  558 144.915 −32.729 75.090 inf inf ATOM  559 O H2O  559 148.122 −32.346 74.911 inf inf ATOM  560 O H2O  560 144.785 −31.977 74.919 inf inf ATOM  561 O H2O  561 144.709 −31.633 74.842 inf inf ATOM  562 O H2O  562 147.630 −31.163 75.079 inf inf ATOM  563 O H2O  563 144.478 −30.848 74.960 inf inf ATOM  564 O H2O  564 144.415 −30.501 74.916 inf inf ATOM  565 O H2O  565 144.093 −30.287 75.119 inf inf ATOM  566 O H2O  566 146.818 −30.130 75.103 inf inf ATOM  567 O H2O  567 146.272 −29.759 74.910 inf inf ATOM  568 O H2O  568 142.960 −29.518 75.103 inf inf ATOM  569 O H2O  569 142.310 −29.100 75.105 inf inf ATOM  570 O H2O  570 142.004 −28.660 75.104 inf inf ATOM  571 O H2O  571 146.664 −28.310 74.901 inf inf ATOM  572 O H2O  572 141.822 −28.102 75.104 inf inf ATOM  573 O H2O  573 141.816 −27.549 75.102 inf inf ATOM  574 O H2O  574 149.400 −27.548 75.104 inf inf ATOM  575 O H2O  575 150.158 −27.593 75.117 inf inf ATOM  576 O H2O  576 148.118 −27.407 75.105 inf inf ATOM  577 O H2O  577 150.390 −27.463 75.138 inf inf ATOM  578 O H2O  578 150.738 −27.013 75.131 inf inf ATOM  579 O H2O  579 142.365 −26.431 75.110 inf inf ATOM  580 O H2O  580 142.493 −26.199 75.126 inf inf ATOM  581 O H2O  581 142.936 −25.693 74.921 inf inf ATOM  582 O H2O  582 151.072 −25.884 75.113 inf inf ATOM  583 O H2O  583 143.613 −25.401 75.153 inf inf ATOM  584 O H2O  584 144.049 −24.964 74.919 inf inf ATOM  585 O H2O  585 150.713 −25.122 75.129 inf inf ATOM  586 O H2O  586 150.310 −24.801 75.086 inf inf ATOM  587 O H2O  587 149.795 −24.215 75.122 inf inf ATOM  588 O H2O  588 149.704 −23.873 75.086 inf inf ATOM  589 O H2O  589 149.724 −23.104 75.098 inf inf ATOM  590 O H2O  590 149.695 −22.390 75.104 inf inf ATOM  591 O H2O  591 144.395 −21.639 75.104 inf inf ATOM  592 O H2O  592 144.381 −21.272 75.104 inf inf ATOM  593 O H2O  593 144.325 −20.875 75.104 inf inf ATOM  594 O H2O  594 148.815 −21.186 75.170 inf inf ATOM  595 O H2O  595 148.056 −20.769 75.125 inf inf ATOM  596 O H2O  596 147.678 −20.390 75.125 inf inf ATOM  597 O H2O  597 147.404 −19.965 75.104 inf inf ATOM  598 O H2O  598 144.005 −19.056 75.085 inf inf ATOM  599 O H2O  599 144.069 −18.647 75.104 inf inf ATOM  600 O H2O  600 147.102 −18.347 75.105 inf inf ATOM  601 O H2O  601 146.996 −18.137 75.104 inf inf ATOM  602 O H2O  602 146.281 −17.548 74.905 inf inf ATOM  603 O H2O  603 145.524 −17.371 75.098 inf inf ATOM  604 O H2O  604 146.818 −34.556 75.447 inf inf ATOM  605 O H2O  605 147.558 −34.572 75.466 inf inf ATOM  606 O H2O  606 146.609 −34.445 75.427 inf inf ATOM  607 O H2O  607 148.090 −34.346 75.497 inf inf ATOM  608 O H2O  608 145.900 −33.998 75.476 inf inf ATOM  609 O H2O  609 145.269 −33.524 75.473 inf inf ATOM  610 O H2O  610 144.975 −33.082 75.473 inf inf ATOM  611 O H2O  611 144.848 −32.746 75.442 inf inf ATOM  612 O H2O  612 144.643 −32.317 75.487 inf inf ATOM  613 O H2O  613 144.495 −32.038 75.427 inf inf ATOM  614 O H2O  614 144.393 −31.614 75.278 inf inf ATOM  615 O H2O  615 144.358 −31.238 75.263 inf inf ATOM  616 O H2O  616 147.519 −30.893 75.485 inf inf ATOM  617 O H2O  617 147.394 −30.674 75.466 inf inf ATOM  618 O H2O  618 147.112 −30.179 75.497 inf inf ATOM  619 O H2O  619 146.848 −29.751 75.452 inf inf ATOM  620 O H2O  620 143.269 −29.654 75.474 inf inf ATOM  621 O H2O  621 142.126 −28.951 75.473 inf inf ATOM  622 O H2O  622 146.785 −29.022 75.504 inf inf ATOM  623 O H2O  623 141.848 −28.328 75.473 inf inf ATOM  624 O H2O  624 141.817 −27.917 75.474 inf inf ATOM  625 O H2O  625 141.838 −27.732 75.473 inf inf ATOM  626 O H2O  626 147.681 −27.640 75.504 inf inf ATOM  627 O H2O  627 142.037 −27.191 75.473 inf inf ATOM  628 O H2O  628 149.223 −27.409 75.500 inf inf ATOM  629 O H2O  629 150.467 −27.153 75.440 inf inf ATOM  630 O H2O  630 150.709 −26.814 75.300 inf inf ATOM  631 O H2O  631 150.755 −26.460 75.350 inf inf ATOM  632 O H2O  632 150.774 −26.070 75.404 inf inf ATOM  633 O H2O  633 150.529 −25.687 75.523 inf inf ATOM  634 O H2O  634 143.895 −25.381 75.457 inf inf ATOM  635 O H2O  635 144.281 −24.995 75.457 inf inf ATOM  636 O H2O  636 150.304 −25.186 75.416 inf inf ATOM  637 O H2O  637 144.572 −24.581 75.482 inf inf ATOM  638 O H2O  638 149.655 −24.267 75.432 inf inf ATOM  639 O H2O  639 149.624 −23.854 75.306 inf inf ATOM  640 O H2O  640 144.748 −23.121 75.471 inf inf ATOM  641 O H2O  641 144.627 −22.368 75.472 inf inf ATOM  642 O H2O  642 144.408 −21.643 75.290 inf inf ATOM  643 O H2O  643 144.410 −21.271 75.448 inf inf ATOM  644 O H2O  644 144.365 −20.899 75.428 inf inf ATOM  645 O H2O  645 144.304 −20.510 75.449 inf inf ATOM  646 O H2O  646 147.562 −20.160 75.471 inf inf ATOM  647 O H2O  647 147.421 −19.839 75.516 inf inf ATOM  648 O H2O  648 147.357 −19.612 75.477 inf inf ATOM  649 O H2O  649 144.035 −18.870 75.473 inf inf ATOM  650 O H2O  650 147.106 −18.345 75.473 inf inf ATOM  651 O H2O  651 144.492 −17.840 75.473 inf inf ATOM  652 O H2O  652 146.662 −17.735 75.473 inf inf ATOM  653 O H2O  653 146.234 −17.479 75.473 inf inf ATOM  654 O H2O  654 146.992 −34.777 75.829 inf inf ATOM  655 O H2O  655 147.949 −34.619 75.826 inf inf ATOM  656 O H2O  656 148.430 −34.154 75.686 inf inf ATOM  657 O H2O  657 145.844 −34.055 75.825 inf inf ATOM  658 O H2O  658 148.787 −33.485 75.779 inf inf ATOM  659 O H2O  659 144.868 −32.773 75.857 inf inf ATOM  660 O H2O  660 148.497 −32.331 75.646 inf inf ATOM  661 O H2O  661 144.392 −31.991 75.654 inf inf ATOM  662 O H2O  662 147.996 −31.560 75.843 inf inf ATOM  663 O H2O  663 144.148 −30.891 75.842 inf inf ATOM  664 O H2O  664 143.945 −30.444 75.842 inf inf ATOM  665 O H2O  665 143.776 −30.184 75.817 inf inf ATOM  666 O H2O  666 143.630 −29.990 75.826 inf inf ATOM  667 O H2O  667 142.958 −29.536 75.842 inf inf ATOM  668 O H2O  668 142.129 −28.950 75.843 inf inf ATOM  669 O H2O  669 141.970 −28.675 75.856 inf inf ATOM  670 O H2O  670 147.153 −28.263 75.853 inf inf ATOM  671 O H2O  671 147.325 −28.056 75.861 inf inf ATOM  672 O H2O  672 147.712 −27.691 75.856 inf inf ATOM  673 O H2O  673 148.476 −27.460 75.818 inf inf ATOM  674 O H2O  674 149.961 −27.191 75.678 inf inf ATOM  675 O H2O  675 142.372 −26.794 75.848 inf inf ATOM  676 O H2O  676 142.584 −26.450 75.650 inf inf ATOM  677 O H2O  677 150.311 −26.440 75.639 inf inf ATOM  678 O H2O  678 150.090 −26.071 75.769 inf inf ATOM  679 O H2O  679 143.513 −25.730 75.833 inf inf ATOM  680 O H2O  680 150.312 −25.707 75.618 inf inf ATOM  681 O H2O  681 144.146 −25.246 75.842 inf inf ATOM  682 O H2O  682 144.326 −25.024 75.842 inf inf ATOM  683 O H2O  683 144.491 −24.830 75.841 inf inf ATOM  684 O H2O  684 144.722 −24.243 75.841 inf inf ATOM  685 O H2O  685 149.443 −23.868 75.861 inf inf ATOM  686 O H2O  686 144.697 −23.119 75.869 inf inf ATOM  687 O H2O  687 149.811 −22.759 75.842 inf inf ATOM  688 O H2O  688 149.741 −22.018 75.846 inf inf ATOM  689 O H2O  689 149.513 −21.561 75.842 inf inf ATOM  690 O H2O  690 149.330 −21.342 75.842 inf inf ATOM  691 O H2O  691 148.497 −20.868 75.653 inf inf ATOM  692 O H2O  692 144.367 −20.488 75.838 inf inf ATOM  693 O H2O  693 148.425 −20.796 75.842 inf inf ATOM  694 O H2O  694 144.320 −19.765 75.813 inf inf ATOM  695 O H2O  695 147.417 −19.590 75.829 inf inf ATOM  696 O H2O  696 144.156 −18.666 75.875 inf inf ATOM  697 O H2O  697 144.426 −17.957 75.656 inf inf ATOM  698 O H2O  698 146.936 −18.177 75.821 inf inf ATOM  699 O H2O  699 146.605 −17.792 75.832 inf inf ATOM  700 O H2O  700 146.213 −17.543 75.793 inf inf ATOM  701 O H2O  701 146.623 −34.767 76.211 inf inf ATOM  702 O H2O  702 148.122 −34.581 76.020 inf inf ATOM  703 O H2O  703 145.815 −34.121 76.211 inf inf ATOM  704 O H2O  704 145.659 −33.855 76.211 inf inf ATOM  705 O H2O  705 148.870 −33.460 76.023 inf inf ATOM  706 O H2O  706 145.207 −33.235 76.194 inf inf ATOM  707 O H2O  707 148.825 −32.721 76.030 inf inf ATOM  708 O H2O  708 148.691 −32.327 76.211 inf inf ATOM  709 O H2O  709 148.344 −31.915 76.201 inf inf ATOM  710 O H2O  710 148.157 −31.744 76.212 inf inf ATOM  711 O H2O  711 147.595 −30.852 76.200 inf inf ATOM  712 O H2O  712 147.486 −30.524 76.233 inf inf ATOM  713 O H2O  713 147.243 −30.093 76.189 inf inf ATOM  714 O H2O  714 143.305 −29.767 76.026 inf inf ATOM  715 O H2O  715 146.980 −29.393 76.031 inf inf ATOM  716 O H2O  716 142.388 −29.022 76.210 inf inf ATOM  717 O H2O  717 142.182 −28.852 76.218 inf inf ATOM  718 O H2O  718 147.259 −28.334 76.162 inf inf ATOM  719 O H2O  719 147.597 −27.958 76.177 inf inf ATOM  720 O H2O  720 148.097 −27.696 76.236 inf inf ATOM  721 O H2O  721 148.836 −27.339 76.212 inf inf ATOM  722 O H2O  722 142.252 −27.043 76.192 inf inf ATOM  723 O H2O  723 149.900 −26.768 75.960 inf inf ATOM  724 O H2O  724 143.063 −26.364 76.249 inf inf ATOM  725 O H2O  725 143.278 −26.032 76.046 inf inf ATOM  726 O H2O  726 149.874 −26.071 75.948 inf inf ATOM  727 O H2O  727 149.486 −25.674 76.261 inf inf ATOM  728 O H2O  728 144.192 −25.304 76.225 inf inf ATOM  729 O H2O  729 149.320 −24.989 76.152 inf inf ATOM  730 O H2O  730 144.672 −24.235 76.231 inf inf ATOM  731 O H2O  731 144.647 −23.480 76.225 inf inf ATOM  732 O H2O  732 149.710 −23.100 76.203 inf inf ATOM  733 O H2O  733 149.803 −22.379 76.220 inf inf ATOM  734 O H2O  734 149.725 −22.023 76.205 inf inf ATOM  735 O H2O  735 144.340 −21.302 76.241 inf inf ATOM  736 O H2O  736 148.651 −20.928 76.211 inf inf ATOM  737 O H2O  737 148.114 −20.525 76.023 inf inf ATOM  738 O H2O  738 144.448 −20.352 76.211 inf inf ATOM  739 O H2O  739 148.028 −20.401 76.212 inf inf ATOM  740 O H2O  740 147.745 −19.977 76.210 inf inf ATOM  741 O H2O  741 147.478 −19.503 76.211 inf inf ATOM  742 O H2O  742 144.293 −18.708 76.171 inf inf ATOM  743 O H2O  743 144.374 −18.290 76.056 inf inf ATOM  744 O H2O  744 146.945 −18.347 75.999 inf inf ATOM  745 O H2O  745 146.641 −17.938 76.034 inf inf ATOM  746 O H2O  746 145.525 −17.677 76.274 inf inf ATOM  747 O H2O  747 146.255 −17.777 76.201 inf inf ATOM  748 O H2O  748 146.623 −34.767 76.581 inf inf ATOM  749 O H2O  749 148.054 −34.664 76.581 inf inf ATOM  750 O H2O  750 148.453 −34.352 76.580 inf inf ATOM  751 O H2O  751 145.414 −33.406 76.555 inf inf ATOM  752 O H2O  752 145.148 −33.088 76.397 inf inf ATOM  753 O H2O  753 145.079 −32.953 76.581 inf inf ATOM  754 O H2O  754 148.690 −32.327 76.581 inf inf ATOM  755 O H2O  755 148.383 −31.887 76.549 inf inf ATOM  756 O H2O  756 148.034 −31.532 76.548 inf inf ATOM  757 O H2O  757 144.168 −30.897 76.599 inf inf ATOM  758 O H2O  758 144.049 −30.684 76.580 inf inf ATOM  759 O H2O  759 143.736 −30.259 76.581 inf inf ATOM  760 O H2O  760 143.396 −29.833 76.580 inf inf ATOM  761 O H2O  761 143.023 −29.443 76.611 inf inf ATOM  762 O H2O  762 142.727 −29.055 76.593 inf inf ATOM  763 O H2O  763 142.348 −28.686 76.620 inf inf ATOM  764 O H2O  764 142.222 −28.465 76.553 inf inf ATOM  765 O H2O  765 142.089 −27.916 76.533 inf inf ATOM  766 O H2O  766 142.120 −27.524 76.455 inf inf ATOM  767 O H2O  767 148.489 −27.567 76.393 inf inf ATOM  768 O H2O  768 142.397 −27.188 76.570 inf inf ATOM  769 O H2O  769 142.597 −26.841 76.377 inf inf ATOM  770 O H2O  770 149.369 −26.777 76.568 inf inf ATOM  771 O H2O  771 149.440 −26.451 76.598 inf inf ATOM  772 O H2O  772 149.554 −26.086 76.381 inf inf ATOM  773 O H2O  773 144.090 −25.559 76.564 inf inf ATOM  774 O H2O  774 144.383 −25.134 76.590 inf inf ATOM  775 O H2O  775 149.180 −24.973 76.379 inf inf ATOM  776 O H2O  776 144.553 −24.225 76.581 inf inf ATOM  777 O H2O  777 149.187 −24.036 76.572 inf inf ATOM  778 O H2O  778 144.436 −23.296 76.593 inf inf ATOM  779 O H2O  779 149.632 −23.071 76.534 inf inf ATOM  780 O H2O  780 144.180 −22.411 76.518 inf inf ATOM  781 O H2O  781 143.935 −22.010 76.652 inf inf ATOM  782 O H2O  782 144.132 −21.640 76.451 inf inf ATOM  783 O H2O  783 144.211 −21.266 76.568 inf inf ATOM  784 O H2O  784 144.246 −20.902 76.588 inf inf ATOM  785 O H2O  785 148.035 −20.455 76.585 inf inf ATOM  786 O H2O  786 144.447 −20.164 76.400 inf inf ATOM  787 O H2O  787 144.455 −19.789 76.575 inf inf ATOM  788 O H2O  788 144.498 −19.380 76.639 inf inf ATOM  789 O H2O  789 147.127 −19.114 76.557 inf inf ATOM  790 O H2O  790 146.773 −18.731 76.537 inf inf ATOM  791 O H2O  791 144.956 −18.299 76.615 inf inf ATOM  792 O H2O  792 144.842 −18.005 76.340 inf inf ATOM  793 O H2O  793 145.525 −17.862 76.483 inf inf ATOM  794 O H2O  794 146.263 −17.949 76.396 inf inf ATOM  795 O H2O  795 146.992 −34.775 76.962 inf inf ATOM  796 O H2O  796 147.948 −34.617 76.966 inf inf ATOM  797 O H2O  797 148.172 −34.471 76.982 inf inf ATOM  798 O H2O  798 145.862 −34.028 76.960 inf inf ATOM  799 O H2O  799 148.818 −33.634 76.949 inf inf ATOM  800 O H2O  800 144.899 −32.766 76.950 inf inf ATOM  801 O H2O  801 144.756 −32.543 76.950 inf inf ATOM  802 O H2O  802 148.483 −31.972 76.764 inf inf ATOM  803 O H2O  803 148.146 −31.577 76.745 inf inf ATOM  804 O H2O  804 148.013 −31.204 76.896 inf inf ATOM  805 O H2O  805 143.937 −30.451 76.950 inf inf ATOM  806 O H2O  806 143.595 −30.064 76.950 inf inf ATOM  807 O H2O  807 143.414 −29.843 76.949 inf inf ATOM  808 O H2O  808 143.066 −29.422 76.951 inf inf ATOM  809 O H2O  809 142.766 −29.017 76.946 inf inf ATOM  810 O H2O  810 142.511 −28.654 76.813 inf inf ATOM  811 O H2O  811 142.289 −28.246 76.666 inf inf ATOM  812 O H2O  812 147.796 −28.495 76.933 inf inf ATOM  813 O H2O  813 148.135 −27.948 76.749 inf inf ATOM  814 O H2O  814 142.551 −27.735 76.967 inf inf ATOM  815 O H2O  815 142.588 −27.190 76.744 inf inf ATOM  816 O H2O  816 142.974 −26.850 76.740 inf inf ATOM  817 O H2O  817 149.258 −26.769 76.927 inf inf ATOM  818 O H2O  818 149.350 −26.425 76.942 inf inf ATOM  819 O H2O  819 143.912 −25.774 76.950 inf inf ATOM  820 O H2O  820 149.159 −25.268 76.950 inf inf ATOM  821 O H2O  821 144.495 −24.937 76.949 inf inf ATOM  822 O H2O  822 144.511 −24.224 76.927 inf inf ATOM  823 O H2O  823 149.125 −23.880 76.977 inf inf ATOM  824 O H2O  824 149.208 −23.668 76.947 inf inf ATOM  825 O H2O  825 149.409 −23.120 76.953 inf inf ATOM  826 O H2O  826 149.465 −22.749 76.993 inf inf ATOM  827 O H2O  827 149.468 −22.379 76.995 inf inf ATOM  828 O H2O  828 143.746 −22.010 76.832 inf inf ATOM  829 O H2O  829 143.784 −21.615 76.871 inf inf ATOM  830 O H2O  830 149.018 −21.285 76.949 inf inf ATOM  831 O H2O  831 148.659 −20.910 76.952 inf inf ATOM  832 O H2O  832 148.105 −20.536 76.766 inf inf ATOM  833 O H2O  833 147.919 −20.166 76.951 inf inf ATOM  834 O H2O  834 147.639 −19.735 76.949 inf inf ATOM  835 O H2O  835 147.450 −19.497 76.982 inf inf ATOM  836 O H2O  836 144.898 −19.008 76.998 inf inf ATOM  837 O H2O  837 144.832 −18.706 76.719 inf inf ATOM  838 O H2O  838 146.438 −18.704 76.938 inf inf ATOM  839 O H2O  839 145.525 −18.298 76.793 inf inf ATOM  840 O H2O  840 146.228 −18.561 76.903 inf inf ATOM  841 O H2O  841 146.812 −34.588 77.327 inf inf ATOM  842 O H2O  842 146.108 −34.161 77.307 inf inf ATOM  843 O H2O  843 148.454 −34.172 77.131 inf inf ATOM  844 O H2O  844 148.536 −34.049 77.317 inf inf ATOM  845 O H2O  845 148.792 −33.615 77.318 inf inf ATOM  846 O H2O  846 148.941 −33.083 77.348 inf inf ATOM  847 O H2O  847 144.635 −32.323 77.319 inf inf ATOM  848 O H2O  848 144.518 −32.012 77.296 inf inf ATOM  849 O H2O  849 144.401 −31.803 77.314 inf inf ATOM  850 O H2O  850 148.131 −31.232 77.124 inf inf ATOM  851 O H2O  851 148.136 −30.877 77.372 inf inf ATOM  852 O H2O  852 148.155 −30.500 77.365 inf inf ATOM  853 O H2O  853 148.121 −30.314 77.312 inf inf ATOM  854 O H2O  854 147.792 −29.736 77.090 inf inf ATOM  855 O H2O  855 147.791 −29.427 77.362 inf inf ATOM  856 O H2O  856 147.740 −29.208 77.319 inf inf ATOM  857 O H2O  857 147.829 −28.619 77.350 inf inf ATOM  858 O H2O  858 142.653 −27.916 77.079 inf inf ATOM  859 O H2O  859 142.874 −27.514 77.192 inf inf ATOM  860 O H2O  860 148.676 −27.561 77.319 inf inf ATOM  861 O H2O  861 149.025 −27.174 77.319 inf inf ATOM  862 O H2O  862 143.372 −26.652 77.299 inf inf ATOM  863 O H2O  863 143.592 −26.217 77.322 inf inf ATOM  864 O H2O  864 143.956 −25.794 77.319 inf inf ATOM  865 O H2O  865 149.164 −25.266 77.318 inf inf ATOM  866 O H2O  866 149.053 −24.958 77.319 inf inf ATOM  867 O H2O  867 149.028 −24.225 77.319 inf inf ATOM  868 O H2O  868 149.073 −23.866 77.320 inf inf ATOM  869 O H2O  869 144.044 −23.306 77.317 inf inf ATOM  870 O H2O  870 149.268 −23.115 77.271 inf inf ATOM  871 O H2O  871 149.314 −22.747 77.287 inf inf ATOM  872 O H2O  872 143.435 −22.010 77.293 inf inf ATOM  873 O H2O  873 149.246 −21.694 77.314 inf inf ATOM  874 O H2O  874 149.163 −21.479 77.317 inf inf ATOM  875 O H2O  875 148.883 −21.062 77.308 inf inf ATOM  876 O H2O  876 148.311 −20.516 77.305 inf inf ATOM  877 O H2O  877 147.989 −20.010 77.300 inf inf ATOM  878 O H2O  878 147.718 −19.813 77.138 inf inf ATOM  879 O H2O  879 147.243 −19.369 77.318 inf inf ATOM  880 O H2O  880 144.989 −19.094 77.300 inf inf ATOM  881 O H2O  881 145.521 −18.664 77.155 inf inf ATOM  882 O H2O  882 146.278 −18.820 77.341 inf inf ATOM  883 O H2O  883 147.002 −34.566 77.680 inf inf ATOM  884 O H2O  884 146.146 −34.124 77.688 inf inf ATOM  885 O H2O  885 148.103 −34.368 77.688 inf inf ATOM  886 O H2O  886 148.519 −34.048 77.688 inf inf ATOM  887 O H2O  887 148.905 −33.411 77.688 inf inf ATOM  888 O H2O  888 144.969 −32.717 77.688 inf inf ATOM  889 O H2O  889 144.771 −32.543 77.686 inf inf ATOM  890 O H2O  890 144.422 −31.974 77.506 inf inf ATOM  891 O H2O  891 148.460 −31.619 77.509 inf inf ATOM  892 O H2O  892 148.409 −31.451 77.688 inf inf ATOM  893 O H2O  893 148.290 −30.869 77.690 inf inf ATOM  894 O H2O  894 143.726 −30.284 77.688 inf inf ATOM  895 O H2O  895 143.405 −29.851 77.688 inf inf ATOM  896 O H2O  896 143.230 −29.658 77.715 inf inf ATOM  897 O H2O  897 142.944 −29.207 77.687 inf inf ATOM  898 O H2O  898 147.962 −28.665 77.672 inf inf ATOM  899 O H2O  899 148.180 −28.214 77.729 inf inf ATOM  900 O H2O  900 143.141 −27.558 77.683 inf inf ATOM  901 O H2O  901 143.263 −27.343 77.701 inf inf ATOM  902 O H2O  902 143.331 −26.822 77.500 inf inf ATOM  903 O H2O  903 143.505 −26.444 77.688 inf inf ATOM  904 O H2O  904 143.757 −25.995 77.688 inf inf ATOM  905 O H2O  905 149.278 −25.703 77.648 inf inf ATOM  906 O H2O  906 144.447 −25.160 77.688 inf inf ATOM  907 O H2O  907 149.065 −24.595 77.679 inf inf ATOM  908 O H2O  908 144.422 −24.038 77.688 inf inf ATOM  909 O H2O  909 143.781 −23.200 77.660 inf inf ATOM  910 O H2O  910 143.469 −22.759 77.681 inf inf ATOM  911 O H2O  911 143.357 −22.413 77.654 inf inf ATOM  912 O H2O  912 143.290 −22.010 77.742 inf inf ATOM  913 O H2O  913 143.320 −21.827 77.691 inf inf ATOM  914 O H2O  914 143.394 −21.246 77.663 inf inf ATOM  915 O H2O  915 148.996 −20.925 77.714 inf inf ATOM  916 O H2O  916 148.691 −20.510 77.659 inf inf ATOM  917 O H2O  917 148.121 −20.150 77.497 inf inf ATOM  918 O H2O  918 144.358 −19.894 77.688 inf inf ATOM  919 O H2O  919 144.793 −19.431 77.502 inf inf ATOM  920 O H2O  920 147.430 −19.491 77.688 inf inf ATOM  921 O H2O  921 145.329 −19.031 77.695 inf inf ATOM  922 O H2O  922 145.894 −18.883 77.685 inf inf ATOM  923 O H2O  923 147.370 −34.566 77.873 inf inf ATOM  924 O H2O  924 146.969 −34.505 78.089 inf inf ATOM  925 O H2O  925 148.303 −34.206 78.062 inf inf ATOM  926 O H2O  926 148.500 −34.029 78.066 inf inf ATOM  927 O H2O  927 148.797 −33.617 78.043 inf inf ATOM  928 O H2O  928 148.960 −33.084 78.047 inf inf ATOM  929 O H2O  929 144.545 −32.402 78.057 inf inf ATOM  930 O H2O  930 144.375 −32.191 78.057 inf inf ATOM  931 O H2O  931 148.552 −31.689 78.057 inf inf ATOM  932 O H2O  932 144.010 −30.818 78.056 inf inf ATOM  933 O H2O  933 148.304 −30.500 78.057 inf inf ATOM  934 O H2O  934 143.461 −29.784 78.068 inf inf ATOM  935 O H2O  935 148.000 −29.374 78.057 inf inf ATOM  936 O H2O  936 147.960 −29.024 78.057 inf inf ATOM  937 O H2O  937 142.914 −28.286 78.004 inf inf ATOM  938 O H2O  938 142.972 −28.105 78.048 inf inf ATOM  939 O H2O  939 143.219 −27.609 78.026 inf inf ATOM  940 O H2O  940 143.423 −27.162 78.074 inf inf ATOM  941 O H2O  941 148.874 −27.010 78.079 inf inf ATOM  942 O H2O  942 149.092 −26.460 78.097 inf inf ATOM  943 O H2O  943 149.229 −26.070 77.878 inf inf ATOM  944 O H2O  944 149.223 −25.701 77.874 inf inf ATOM  945 O H2O  945 149.226 −25.330 77.871 inf inf ATOM  946 O H2O  946 149.183 −24.972 77.888 inf inf ATOM  947 O H2O  947 149.286 −24.589 78.141 inf inf ATOM  948 O H2O  948 144.333 −23.791 78.057 inf inf ATOM  949 O H2O  949 149.262 −23.856 78.104 inf inf ATOM  950 O H2O  950 143.757 −23.223 78.057 inf inf ATOM  951 O H2O  951 149.141 −23.090 78.057 inf inf ATOM  952 O H2O  952 143.324 −22.422 78.057 inf inf ATOM  953 O H2O  953 149.200 −22.010 77.874 inf inf ATOM  954 O H2O  954 143.308 −21.640 78.058 inf inf ATOM  955 O H2O  955 143.323 −21.229 78.057 inf inf ATOM  956 O H2O  956 149.141 −20.856 77.994 inf inf ATOM  957 O H2O  957 148.869 −20.515 77.859 inf inf ATOM  958 O H2O  958 143.903 −20.204 78.057 inf inf ATOM  959 O H2O  959 144.102 −20.061 78.057 inf inf ATOM  960 O H2O  960 148.140 −19.906 78.036 inf inf ATOM  961 O H2O  961 147.217 −19.379 78.073 inf inf ATOM  962 O H2O  962 145.351 −19.082 78.050 inf inf ATOM  963 O H2O  963 145.894 −18.942 78.047 inf inf ATOM  964 O H2O  964 146.655 −34.309 78.403 inf inf ATOM  965 O H2O  965 148.036 −34.269 78.391 inf inf ATOM  966 O H2O  966 148.429 −33.959 78.407 inf inf ATOM  967 O H2O  967 148.825 −33.447 78.232 inf inf ATOM  968 O H2O  968 148.820 −33.265 78.411 inf inf ATOM  969 O H2O  969 148.872 −32.715 78.373 inf inf ATOM  970 O H2O  970 148.828 −32.533 78.416 inf inf ATOM  971 O H2O  971 148.613 −32.015 78.397 inf inf ATOM  972 O H2O  972 148.486 −31.605 78.247 inf inf ATOM  973 O H2O  973 144.070 −31.051 78.426 inf inf ATOM  974 O H2O  974 143.705 −30.118 78.229 inf inf ATOM  975 O H2O  975 143.713 −29.935 78.403 inf inf ATOM  976 O H2O  976 147.955 −29.384 78.440 inf inf ATOM  977 O H2O  977 147.914 −29.024 78.422 inf inf ATOM  978 O H2O  978 143.007 −28.305 78.213 inf inf ATOM  979 O H2O  979 148.068 −28.084 78.427 inf inf ATOM  980 O H2O  980 143.495 −27.547 78.425 inf inf ATOM  981 O H2O  981 148.482 −27.365 78.428 inf inf ATOM  982 O H2O  982 148.704 −26.823 78.455 inf inf ATOM  983 O H2O  983 148.825 −26.617 78.412 inf inf ATOM  984 O H2O  984 144.087 −25.935 78.397 inf inf ATOM  985 O H2O  985 144.723 −25.275 78.337 inf inf ATOM  986 O H2O  986 149.296 −25.296 78.429 inf inf ATOM  987 O H2O  987 144.803 −24.593 78.216 inf inf ATOM  988 O H2O  988 144.966 −24.227 78.431 inf inf ATOM  989 O H2O  989 144.615 −23.837 78.404 inf inf ATOM  990 O H2O  990 144.418 −23.670 78.426 inf inf ATOM  991 O H2O  991 143.799 −23.182 78.448 inf inf ATOM  992 O H2O  992 143.625 −22.969 78.444 inf inf ATOM  993 O H2O  993 143.299 −22.010 78.243 inf inf ATOM  994 O H2O  994 143.304 −21.641 78.243 inf inf ATOM  995 O H2O  995 149.378 −21.275 78.433 inf inf ATOM  996 O H2O  996 143.577 −20.589 78.398 inf inf ATOM  997 O H2O  997 143.908 −20.209 78.408 inf inf ATOM  998 O H2O  998 144.390 −19.918 78.444 inf inf ATOM  999 O H2O  999 148.275 −19.838 78.439 inf inf ATOM 1000 O H2O 1000 147.386 −19.574 78.437 inf inf ATOM 1001 O H2O 1001 145.525 −19.060 78.240 inf inf ATOM 1002 O H2O 1002 146.298 −19.104 78.495 inf inf ATOM 1003 O H2O 1003 146.946 −19.324 78.399 inf inf ATOM 1004 O H2O 1004 147.370 −34.263 78.720 inf inf ATOM 1005 O H2O 1005 148.079 −34.133 78.582 inf inf ATOM 1006 O H2O 1006 146.972 −34.095 78.883 inf inf ATOM 1007 O H2O 1007 148.274 −33.804 78.776 inf inf ATOM 1008 O H2O 1008 148.437 −33.316 78.763 inf inf ATOM 1009 O H2O 1009 145.901 −33.261 78.790 inf inf ATOM 1010 O H2O 1010 145.204 −32.755 78.747 inf inf ATOM 1011 O H2O 1011 144.613 −32.334 78.791 inf inf ATOM 1012 O H2O 1012 144.452 −32.138 78.784 inf inf ATOM 1013 O H2O 1013 144.224 −31.610 78.799 inf inf ATOM 1014 O H2O 1014 144.082 −30.869 78.588 inf inf ATOM 1015 O H2O 1015 144.159 −30.522 78.827 inf inf ATOM 1016 O H2O 1016 148.062 −30.097 78.864 inf inf ATOM 1017 O H2O 1017 143.889 −29.753 78.778 inf inf ATOM 1018 O H2O 1018 143.845 −29.401 78.815 inf inf ATOM 1019 O H2O 1019 143.691 −29.202 78.777 inf inf ATOM 1020 O H2O 1020 147.810 −28.622 78.757 inf inf ATOM 1021 O H2O 1021 143.377 −27.943 78.544 inf inf ATOM 1022 O H2O 1022 148.024 −28.046 78.767 inf inf ATOM 1023 O H2O 1023 148.327 −27.562 78.805 inf inf ATOM 1024 O H2O 1024 148.431 −27.213 78.830 inf inf ATOM 1025 O H2O 1025 148.565 −26.776 78.761 inf inf ATOM 1026 O H2O 1026 148.805 −26.419 78.590 inf inf ATOM 1027 O H2O 1027 148.782 −26.223 78.763 inf inf ATOM 1028 O H2O 1028 144.785 −25.884 78.797 inf inf ATOM 1029 O H2O 1029 145.144 −25.326 78.628 inf inf ATOM 1030 O H2O 1030 149.355 −25.316 78.810 inf inf ATOM 1031 O H2O 1031 145.194 −24.592 78.573 inf inf ATOM 1032 O H2O 1032 145.314 −24.234 78.813 inf inf ATOM 1033 O H2O 1033 145.184 −24.023 78.773 inf inf ATOM 1034 O H2O 1034 144.804 −23.642 78.773 inf inf ATOM 1035 O H2O 1035 144.387 −23.391 78.855 inf inf ATOM 1036 O H2O 1036 143.727 −22.909 78.771 inf inf ATOM 1037 O H2O 1037 149.224 −22.381 78.610 inf inf ATOM 1038 O H2O 1038 149.312 −21.997 78.795 inf inf ATOM 1039 O H2O 1039 149.393 −21.272 78.795 inf inf ATOM 1040 O H2O 1040 143.642 −20.694 78.808 inf inf ATOM 1041 O H2O 1041 144.048 −20.165 78.611 inf inf ATOM 1042 O H2O 1042 148.936 −20.259 78.795 inf inf ATOM 1043 O H2O 1043 144.950 −19.764 78.816 inf inf ATOM 1044 O H2O 1044 145.150 −19.604 78.802 inf inf ATOM 1045 O H2O 1045 146.986 −19.456 78.596 inf inf ATOM 1046 O H2O 1046 145.549 −19.317 78.745 inf inf ATOM 1047 O H2O 1047 146.600 −19.338 78.732 inf inf ATOM 1048 O H2O 1048 147.370 −33.905 79.095 inf inf ATOM 1049 O H2O 1049 146.466 −33.434 79.126 inf inf ATOM 1050 O H2O 1050 147.747 −33.654 79.189 inf inf ATOM 1051 O H2O 1051 145.890 −33.089 78.985 inf inf ATOM 1052 O H2O 1052 148.270 −33.084 79.130 inf inf ATOM 1053 O H2O 1053 145.678 −32.761 79.195 inf inf ATOM 1054 O H2O 1054 144.772 −32.364 78.991 inf inf ATOM 1055 O H2O 1055 148.224 −32.373 79.096 inf inf ATOM 1056 O H2O 1056 144.758 −32.190 79.187 inf inf ATOM 1057 O H2O 1057 144.352 −31.567 79.085 inf inf ATOM 1058 O H2O 1058 144.326 −31.238 79.102 inf inf ATOM 1059 O H2O 1059 147.917 −30.869 79.160 inf inf ATOM 1060 O H2O 1060 144.523 −30.499 79.242 inf inf ATOM 1061 O H2O 1061 144.285 −30.110 79.112 inf inf ATOM 1062 O H2O 1062 144.244 −29.762 79.153 inf inf ATOM 1063 O H2O 1063 144.049 −29.393 78.978 inf inf ATOM 1064 O H2O 1064 143.743 −28.992 78.885 inf inf ATOM 1065 O H2O 1065 147.666 −29.023 78.907 inf inf ATOM 1066 O H2O 1066 144.421 −28.841 79.149 inf inf ATOM 1067 O H2O 1067 143.719 −28.306 78.920 inf inf ATOM 1068 O H2O 1068 147.613 −28.333 79.223 inf inf ATOM 1069 O H2O 1069 144.614 −27.916 79.137 inf inf ATOM 1070 O H2O 1070 144.082 −27.547 78.929 inf inf ATOM 1071 O H2O 1071 148.239 −27.529 79.146 inf inf ATOM 1072 O H2O 1072 148.329 −27.188 79.180 inf inf ATOM 1073 O H2O 1073 148.470 −26.807 78.977 inf inf ATOM 1074 O H2O 1074 144.784 −26.628 79.192 inf inf ATOM 1075 O H2O 1075 144.471 −26.142 78.891 inf inf ATOM 1076 O H2O 1076 145.349 −26.076 79.143 inf inf ATOM 1077 O H2O 1077 145.462 −25.652 79.042 inf inf ATOM 1078 O H2O 1078 149.042 −25.714 79.167 inf inf ATOM 1079 O H2O 1079 149.378 −25.317 79.165 inf inf ATOM 1080 O H2O 1080 145.551 −24.583 78.954 inf inf ATOM 1081 O H2O 1081 149.589 −24.593 79.164 inf inf ATOM 1082 O H2O 1082 149.592 −24.408 79.165 inf inf ATOM 1083 O H2O 1083 149.471 −23.838 79.165 inf inf ATOM 1084 O H2O 1084 149.364 −23.497 79.159 inf inf ATOM 1085 O H2O 1085 144.762 −23.337 79.188 inf inf ATOM 1086 O H2O 1086 144.067 −22.919 79.141 inf inf ATOM 1087 O H2O 1087 143.827 −22.393 79.201 inf inf ATOM 1088 O H2O 1088 143.786 −22.037 79.242 inf inf ATOM 1089 O H2O 1089 149.331 −21.640 79.142 inf inf ATOM 1090 O H2O 1090 143.684 −21.089 79.161 inf inf ATOM 1091 O H2O 1091 149.056 −20.517 79.173 inf inf ATOM 1092 O H2O 1092 144.397 −20.299 79.214 inf inf ATOM 1093 O H2O 1093 144.814 −20.072 79.106 inf inf ATOM 1094 O H2O 1094 145.474 −19.728 79.097 inf inf ATOM 1095 O H2O 1095 147.024 −19.709 79.016 inf inf ATOM 1096 O H2O 1096 147.923 −19.802 79.162 inf inf ATOM 1097 O H2O 1097 145.546 −19.480 78.926 inf inf ATOM 1098 O H2O 1098 146.263 −19.436 78.970 inf inf ATOM 1099 O H2O 1099 146.670 −33.415 79.234 inf inf ATOM 1100 O H2O 1100 146.290 −33.048 79.304 inf inf ATOM 1101 O H2O 1101 147.746 −33.091 79.376 inf inf ATOM 1102 O H2O 1102 146.287 −32.839 79.474 inf inf ATOM 1103 O H2O 1103 147.405 −32.750 79.489 inf inf ATOM 1104 O H2O 1104 145.497 −32.403 79.433 inf inf ATOM 1105 O H2O 1105 147.562 −32.349 79.544 inf inf ATOM 1106 O H2O 1106 144.977 −31.971 79.521 inf inf ATOM 1107 O H2O 1107 147.830 −31.977 79.440 inf inf ATOM 1108 O H2O 1108 147.618 −31.608 79.623 inf inf ATOM 1109 O H2O 1109 147.574 −31.229 79.563 inf inf ATOM 1110 O H2O 1110 147.546 −30.870 79.520 inf inf ATOM 1111 O H2O 1111 144.944 −30.499 79.596 inf inf ATOM 1112 O H2O 1112 144.703 −30.131 79.432 inf inf ATOM 1113 O H2O 1113 144.467 −29.762 79.274 inf inf ATOM 1114 O H2O 1114 147.355 −29.954 79.512 inf inf ATOM 1115 O H2O 1115 144.932 −29.374 79.592 inf inf ATOM 1116 O H2O 1116 147.343 −29.420 79.270 inf inf ATOM 1117 O H2O 1117 146.780 −29.024 79.478 inf inf ATOM 1118 O H2O 1118 145.106 −28.633 79.423 inf inf ATOM 1119 O H2O 1119 146.632 −28.840 79.528 inf inf ATOM 1120 O H2O 1120 144.827 −28.284 79.236 inf inf ATOM 1121 O H2O 1121 146.984 −28.453 79.499 inf inf ATOM 1122 O H2O 1122 145.147 −27.904 79.390 inf inf ATOM 1123 O H2O 1123 147.366 −28.093 79.525 inf inf ATOM 1124 O H2O 1124 145.158 −27.547 79.336 inf inf ATOM 1125 O H2O 1125 148.115 −27.551 79.353 inf inf ATOM 1126 O H2O 1126 145.449 −27.145 79.458 inf inf ATOM 1127 O H2O 1127 144.802 −26.808 79.181 inf inf ATOM 1128 O H2O 1128 148.228 −26.786 79.490 inf inf ATOM 1129 O H2O 1129 148.431 −26.417 79.326 inf inf ATOM 1130 O H2O 1130 148.451 −26.238 79.525 inf inf ATOM 1131 O H2O 1131 146.015 −25.703 79.596 inf inf ATOM 1132 O H2O 1132 145.967 −25.331 79.614 inf inf ATOM 1133 O H2O 1133 145.954 −24.963 79.565 inf inf ATOM 1134 O H2O 1134 149.578 −24.594 79.348 inf inf ATOM 1135 O H2O 1135 149.516 −24.196 79.563 inf inf ATOM 1136 O H2O 1136 145.102 −23.522 79.385 inf inf ATOM 1137 O H2O 1137 144.750 −23.173 79.386 inf inf ATOM 1138 O H2O 1138 149.221 −23.301 79.535 inf inf ATOM 1139 O H2O 1139 144.604 −22.746 79.531 inf inf ATOM 1140 O H2O 1140 144.246 −22.370 79.501 inf inf ATOM 1141 O H2O 1141 143.993 −22.038 79.431 inf inf ATOM 1142 O H2O 1142 143.732 −21.641 79.296 inf inf ATOM 1143 O H2O 1143 149.254 −21.641 79.365 inf inf ATOM 1144 O H2O 1144 149.257 −21.272 79.367 inf inf ATOM 1145 O H2O 1145 149.066 −20.891 79.557 inf inf ATOM 1146 O H2O 1146 144.408 −20.708 79.553 inf inf ATOM 1147 O H2O 1147 145.524 −20.457 79.457 inf inf ATOM 1148 O H2O 1148 144.786 −20.414 79.469 inf inf ATOM 1149 O H2O 1149 145.893 −20.163 79.352 inf inf ATOM 1150 O H2O 1150 146.630 −20.338 79.547 inf inf ATOM 1151 O H2O 1151 148.803 −20.392 79.491 inf inf ATOM 1152 O H2O 1152 147.022 −20.033 79.480 inf inf ATOM 1153 O H2O 1153 148.104 −19.992 79.525 inf inf ATOM 1154 O H2O 1154 147.001 −32.658 79.633 inf inf ATOM 1155 O H2O 1155 147.015 −32.366 79.766 inf inf ATOM 1156 O H2O 1156 145.879 −31.999 79.770 inf inf ATOM 1157 O H2O 1157 146.817 −31.979 79.910 inf inf ATOM 1158 O H2O 1158 145.145 −31.615 79.743 inf inf ATOM 1159 O H2O 1159 146.446 −31.608 79.913 inf inf ATOM 1160 O H2O 1160 144.843 −31.238 79.633 inf inf ATOM 1161 O H2O 1161 145.894 −31.422 79.898 inf inf ATOM 1162 O H2O 1162 146.632 −31.424 79.899 inf inf ATOM 1163 O H2O 1163 145.144 −30.861 79.746 inf inf ATOM 1164 O H2O 1164 146.263 −30.842 79.826 inf inf ATOM 1165 O H2O 1165 145.162 −30.500 79.703 inf inf ATOM 1166 O H2O 1166 146.632 −30.500 79.769 inf inf ATOM 1167 O H2O 1167 145.502 −30.131 79.810 inf inf ATOM 1168 O H2O 1168 146.994 −30.135 79.693 inf inf ATOM 1169 O H2O 1169 145.894 −29.762 79.860 inf inf ATOM 1170 O H2O 1170 145.159 −29.395 79.710 inf inf ATOM 1171 O H2O 1171 146.584 −29.417 79.648 inf inf ATOM 1172 O H2O 1172 146.232 −29.048 79.617 inf inf ATOM 1173 O H2O 1173 146.223 −28.646 79.584 inf inf ATOM 1174 O H2O 1174 146.633 −28.245 79.609 inf inf ATOM 1175 O H2O 1175 146.631 −27.946 79.762 inf inf ATOM 1176 O H2O 1176 146.231 −27.613 79.851 inf inf ATOM 1177 O H2O 1177 147.356 −27.702 79.859 inf inf ATOM 1178 O H2O 1178 146.074 −27.180 79.909 inf inf ATOM 1179 O H2O 1179 148.034 −27.148 79.643 inf inf ATOM 1180 O H2O 1180 147.887 −26.790 79.848 inf inf ATOM 1181 O H2O 1181 147.927 −26.441 79.908 inf inf ATOM 1182 O H2O 1182 146.261 −26.254 79.908 inf inf ATOM 1183 O H2O 1183 148.287 −26.063 79.894 inf inf ATOM 1184 O H2O 1184 148.880 −25.742 79.739 inf inf ATOM 1185 O H2O 1185 148.914 −25.584 79.957 inf inf ATOM 1186 O H2O 1186 149.250 −25.162 79.923 inf inf ATOM 1187 O H2O 1187 145.774 −24.206 79.894 inf inf ATOM 1188 O H2O 1188 145.644 −23.893 79.912 inf inf ATOM 1189 O H2O 1189 149.162 −23.453 79.936 inf inf ATOM 1190 O H2O 1190 145.150 −23.307 79.904 inf inf ATOM 1191 O H2O 1191 149.120 −22.748 79.903 inf inf ATOM 1192 O H2O 1192 149.115 −22.379 79.903 inf inf ATOM 1193 O H2O 1193 149.050 −22.004 79.911 inf inf ATOM 1194 O H2O 1194 144.786 −21.825 79.903 inf inf ATOM 1195 O H2O 1195 144.404 −21.263 79.748 inf inf ATOM 1196 O H2O 1196 144.435 −20.921 79.663 inf inf ATOM 1197 O H2O 1197 145.508 −21.056 79.948 inf inf ATOM 1198 O H2O 1198 148.851 −21.086 79.908 inf inf ATOM 1199 O H2O 1199 145.921 −20.597 79.655 inf inf ATOM 1200 O H2O 1200 146.624 −20.663 79.934 inf inf ATOM 1201 O H2O 1201 148.805 −20.575 79.677 inf inf ATOM 1202 O H2O 1202 147.364 −20.127 79.749 inf inf ATOM 1203 O H2O 1203 148.117 −20.139 79.735 inf inf ATOM 1204 O H2O 1204 146.632 −31.608 79.925 inf inf ATOM 1205 O H2O 1205 147.330 −27.507 79.967 inf inf ATOM 1206 O H2O 1206 147.360 −27.173 80.053 inf inf ATOM 1207 O H2O 1207 147.366 −26.809 80.072 inf inf ATOM 1208 O H2O 1208 147.001 −26.440 80.108 inf inf ATOM 1209 O H2O 1209 147.001 −26.089 80.155 inf inf ATOM 1210 O H2O 1210 146.620 −25.707 80.106 inf inf ATOM 1211 O H2O 1211 147.736 −25.880 80.263 inf inf ATOM 1212 O H2O 1212 146.311 −25.313 80.039 inf inf ATOM 1213 O H2O 1213 148.793 −25.463 80.218 inf inf ATOM 1214 O H2O 1214 149.225 −24.966 80.093 inf inf ATOM 1215 O H2O 1215 149.102 −24.594 80.343 inf inf ATOM 1216 O H2O 1216 149.096 −24.225 80.337 inf inf ATOM 1217 O H2O 1217 149.196 −23.862 80.074 inf inf ATOM 1218 O H2O 1218 145.135 −23.127 80.098 inf inf ATOM 1219 O H2O 1219 145.162 −22.931 80.268 inf inf ATOM 1220 O H2O 1220 144.849 −22.043 79.987 inf inf ATOM 1221 O H2O 1221 148.947 −22.038 80.215 inf inf ATOM 1222 O H2O 1222 148.825 −21.839 80.254 inf inf ATOM 1223 O H2O 1223 145.522 −21.455 80.275 inf inf ATOM 1224 O H2O 1224 148.543 −21.223 80.202 inf inf ATOM 1225 O H2O 1225 146.236 −21.008 80.324 inf inf ATOM 1226 O H2O 1226 148.492 −20.896 80.108 inf inf ATOM 1227 O H2O 1227 147.370 −20.747 80.228 inf inf ATOM 1228 O H2O 1228 148.090 −20.746 80.207 inf inf ATOM 1229 O H2O 1229 147.369 −25.639 80.362 inf inf ATOM 1230 O H2O 1230 146.992 −25.350 80.483 inf inf ATOM 1231 O H2O 1231 148.489 −25.355 80.491 inf inf ATOM 1232 O H2O 1232 146.999 −25.152 80.645 inf inf ATOM 1233 O H2O 1233 148.467 −25.137 80.619 inf inf ATOM 1234 O H2O 1234 146.318 −24.710 80.600 inf inf ATOM 1235 O H2O 1235 145.894 −24.225 80.456 inf inf ATOM 1236 O H2O 1236 145.821 −23.819 80.604 inf inf ATOM 1237 O H2O 1237 145.558 −23.461 80.437 inf inf ATOM 1238 O H2O 1238 145.673 −23.136 80.695 inf inf ATOM 1239 O H2O 1239 145.213 −22.748 80.418 inf inf ATOM 1240 O H2O 1240 148.880 −22.748 80.480 inf inf ATOM 1241 O H2O 1241 148.877 −22.379 80.477 inf inf ATOM 1242 O H2O 1242 148.632 −22.022 80.611 inf inf ATOM 1243 O H2O 1243 148.288 −21.647 80.633 inf inf ATOM 1244 O H2O 1244 145.907 −21.466 80.624 inf inf ATOM 1245 O H2O 1245 147.398 −21.200 80.559 inf inf ATOM 1246 O H2O 1246 148.109 −21.271 80.458 inf inf ATOM 1247 O H2O 1247 147.001 −21.124 80.604 inf inf ATOM 1248 O H2O 1248 148.062 −20.949 80.316 inf inf ATOM 1249 O H2O 1249 148.108 −24.957 80.817 inf inf ATOM 1250 O H2O 1250 146.487 −24.554 80.971 inf inf ATOM 1251 O H2O 1251 148.087 −24.736 80.946 inf inf ATOM 1252 O H2O 1252 146.304 −24.378 80.959 inf inf ATOM 1253 O H2O 1253 146.090 −23.851 80.999 inf inf ATOM 1254 O H2O 1254 146.076 −23.487 81.012 inf inf ATOM 1255 O H2O 1255 146.081 −23.117 81.006 inf inf ATOM 1256 O H2O 1256 146.081 −22.748 81.003 inf inf ATOM 1257 O H2O 1257 145.568 −22.379 80.760 inf inf ATOM 1258 O H2O 1258 148.489 −22.379 80.843 inf inf ATOM 1259 O H2O 1259 148.106 −22.196 81.002 inf inf ATOM 1260 O H2O 1260 146.242 −21.599 80.888 inf inf ATOM 1261 O H2O 1261 147.014 −21.806 81.056 inf inf ATOM 1262 O H2O 1262 147.739 −21.845 80.964 inf inf ATOM 1263 O H2O 1263 146.632 −21.300 80.783 inf inf ATOM 1264 O H2O 1264 147.370 −24.619 81.232 inf inf ATOM 1265 O H2O 1265 146.595 −24.262 81.250 inf inf ATOM 1266 O H2O 1266 147.727 −24.385 81.343 inf inf ATOM 1267 O H2O 1267 146.275 −23.851 81.183 inf inf ATOM 1268 O H2O 1268 148.255 −23.836 81.322 inf inf ATOM 1269 O H2O 1269 148.264 −23.487 81.336 inf inf ATOM 1270 O H2O 1270 146.617 −23.298 81.405 inf inf ATOM 1271 O H2O 1271 148.173 −23.069 81.308 inf inf ATOM 1272 O H2O 1272 147.002 −22.681 81.331 inf inf ATOM 1273 O H2O 1273 147.751 −22.915 81.420 inf inf ATOM 1274 O H2O 1274 146.632 −22.380 81.191 inf inf ATOM 1275 O H2O 1275 147.728 −22.392 81.160 inf inf ATOM 1276 O H2O 1276 147.001 −22.020 81.123 inf inf ATOM 1277 O H2O 1277 147.370 −24.195 81.476 inf inf ATOM 1278 O H2O 1278 147.370 −23.858 81.573 inf inf ATOM 1279 O H2O 1279 147.002 −23.487 81.559 inf inf ATOM 1280 O H2O 1280 147.009 −23.134 81.506 inf inf 

We claim:
 1. A compound of the formula:


2. A compound of the formula:


3. A compound of the formula:


4. A compound of the formula:


5. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula:

and a pharmaceutically acceptable carrier or diluent.
 6. A method comprising contacting HIV reverse transcriptase with an inhibitory effective amount of a compound of the formula;


7. A method comprising administering to a subject an anti-retrovirus effective amount of a compound of the formula:


8. A method comprising contacting a retrovirus with a retrovirus replication inhibitory amount of a compound of the formula;


9. A pharmaceutical composition comprising an anti-retrovirus effective amount of a compound of the formula:

and a pharmaceutically acceptable carrier.
 10. A pharmaceutical composition comprising an HIV inhibitory effective amount of a compound of the formula:

and a pharmaceutically acceptable carrier.
 11. A pharmaceutical composition comprising an AIDS treating effective amount of a compound of the formula:

and a pharmaceutically acceptable carrier.
 12. A pharmaceutical composition comprising an HIV reverse transcriptase inhibitory effective amount of a compound of the formula:

and a pharmaceutically acceptable carrier.
 13. A composition comprising a retrovirus replication inhibiting amount of a compound of the formula:

and a pharmaceutically acceptable carrier. 